CN101283280A - Protein markers for diagnosing if colorectal cancer and use of said markers as drug targets for the treatment of said cance type - Google Patents

Abstract

This invention relates to a method for diagnosing colorectal cancer (CRC) in a mammal, such as a human, the method comprising determining the presence, absence or level of expression of at least one marker protein in a biological sample from said mammal. It is demonstrated that the combination of metabolic labelling of colon biopsies from both normal and tumour tissues followed by 2 dimensional gel electrophoresis and mass spectrometry is a powerful tool in the identification of marker proteins involved in the development of CRC.

Description

Be used to diagnose the protein labeling of colorectal cancer and described mark purposes as the drug target of described cancer types treatment

Background of invention

The present invention relates generally to the protein with the colorectal cancer correlated expression, identifies this method of protein, the method for screening of medicaments, this protein expression of this drug influence or activity, and the therapeutic compound that is used for the treatment of and prevents colorectal cancer.

Background of invention

Colorectal cancer (CRC) is the third-largest cancer types among developed country such as the US, and is the reason of second largest cancer associated death.For masculinity and femininity, estimated that the lifelong risk that produces CRC is near 6%.For screening, CRC is suitable disease, because it is to have long asymptomatic preclinical common malignant disease, and if find to have high viability in early days.Because the evidence of accumulation shows that consumingly cancer is caused by so-called adenoma, can obtain the prevention of CRC by the screening sequence that detects adenoma and cause removing them.

Colorectal cancer (CRC) is the term that is used for being illustrated in the cancer that produces in colon or the rectum.Colon and rectum are the parts of digestive system, and digestive system is also referred to as intestines and stomach or GI system.Digestive system is handled the major part (fecal matter or ight soil) that food is used for energy and removes solid waste.Food arrives stomach by esophagus after chewing and swallowing.Food obtains part under one's belt and decomposes, and is sent to small intestine then, is also referred to as little enteron aisle.Word " little " refers to the diameter of small intestine, and its diameter than large intestine is narrow.In fact, small intestine is the longest part of digestive system.Small intestine continues to decompose food and absorb most nutrient.Small intestine connects colon at the bottom right abdomen.Colon is divided into three parts: (right side) of rising, horizontal and (left side) that descend.Colon tehn connects rectum at left lower quadrant.The function of colon is continue to absorb water and mineral matter and as the storage place of refuse from food.The refuse that stays after this process is an ight soil, and enters in the rectum, and rectum is last part of digestive system.Ight soil excretes by anus from rectum.

The wall of colon and rectum has several layer tissues.CRC begins also can pass through some or all other layer growths at innermost layer.Know that some knowledge about these layers are important, have how deeply because the stage of colorectal cancer (diffusion) is depended on these layers of invasion to a great extent.The colon cancer and the carcinoma of the rectum always are called colorectal cancer (CRC), have many common features, and discuss together at this.In most of people, colorectal cancer is slowly development in the time period of several years.Before real cancer produced, tissue or growth of tumor began as non-cancer polyp usually, and it finally becomes cancer.Polyp produces on the internal layer of colon or rectum.The polyp of particular types is called adenoma polyp or adenoma, has the potential that becomes cancer.The polyp that has other types is called hyperplasia and inflammatory polyp.Usually do not think that inflammatory polyp and hyperplastic polyp are before the cancer.But some doctors think that some hyperplastic polyps may be the signals that preceding pathology of cancer or people more may produce adenoma polyp and cancer, if particularly they grow in the right side or the colon ascendens.Situation is called dysplasia before the another kind of cancer.This sees that ulcerative colitis causes the chronic inflammation of colon usually in ill crowd as ulcerative colitis.

In case form cancer in polyp, it finally begins to grow in the wall of colon or rectum.In case they are in wall, cancer cell can grow in blood vessel or the lymphatic vessel.Lymphatic vessel is the minim channel that approaches, and takes away and shifts out EV extracellular fluid body and leucocyte.In near they at first arrange into the lymph node, lymph node is to help anti-infectious bean shaped structure.When cancer cell diffused in the blood vessel, they can move to the distal part of health.This diffusion process is called transfer.Colorectal cancer above 95% is a gland cancer.These are the cancers that are arranged in the gland cell of colon and rectal wall internal layer.In colon and rectum, also may produce other not too tumours of common type, as:

The specialized hormone that carcinoid--these cancers result from intestines produces cell.

GISTs--these tumours result from the colon wall the specialized cell that is called " interstitial cell of Cajal ".Some be optimum (non-cancer); Other be pernicious (cancer).Although these cancers can find that Anywhere they are usually in colon GI.

Lymphadenoma--these are cancers of immune system cell, produce in lymph node usually, but also can start from colon and rectum or other organs.

Usually in one of three kinds of AD HOC, observe CRC: distribute, heredity or family.

Distribute disease, do not have the inducement of family or heredity, occupy about 70% of colorectal cancer among the crowd.Distributing colon cancer or CRC is common in greater than 50 years old people, may be the result of meals and environmental factor and unaccelerated aging.

Be lower than the hereditary inducement that 10% patient has colon cancer.The symptom of heredity comprise polyp of colon wherein be disease mainly show those and wherein they no those.The polyp disease symptoms is subdivided into the sick and paramnesia polyposis symptom of familial adenomatous polyposis.The cardinal symptom of non-polyposis comprises the non-polyposis colorectal cancer (HNPCC) (Lynch syndrome I) and the cancer family syndrome (Lynch syndrome II) of heredity.Although uncommon, these syndromes provide the understanding to all colorectal cancer types of biological.

The 3rd and the CRC generation pattern of minimum understanding be considered to familial colon carcinoma.In affected family, it is too high so that can not think the colon cancer of distributing that colon cancer produces frequency, and be not and the corresponding to pattern of the symptom of heredity.All colon cancer cases up to 25% can belong to this classification.

Think that the molecular basis that distributes colon cancer follows the rapid model of multistep of carcinogenesis.This model description the accumulation of hereditary situation, each situation gives optionally growth vigor for affected colon cell.These changes finally cause uncontrolled cell growth, and propagation and asexual tumour produce.The accumulative effect of these somatic mutations is the inducements of distributing CRC.Obtain four main conclusions from this model:

1) colorectal cancer is caused by the sudden change activation of oncogene and the deactivation of tumor suppressor gene;

2) vicious transformation needs the somatic mutation of at least four or five genes of cell.

3) order of the accumulation of many genetic mutations rather than sudden change has determined the biological behavior of tumour, although the sudden change of apc gene usually occurs in the early stage of process, and the sudden change of p53 suppressor usually occurs in the late period of process; With

4) feature of the oncogenic process of colon cancer is applicable to other entity tumors, as breast cancer and cancer of pancreas.

The colon cancer syndrome of normal heredity is familial adenomatous polyposis disease and HNPCC.In these syndromes each is the result of specific germ line mutation.In the familial adenomatous polyposis disease, germ line mutation is apc gene normally, a kind of tumor suppressor gene.

In HNPCC, a kind of in the MMR gene produced sudden change, and modal is hMLH1 or hMSH2.Several in the paramnesia polyp syndrome are associated with germ line mutation recently.An example is the Peutz-Jeghers syndrome, and it is caused unusually by the STK11 tumor suppressor gene.

The screening sequence of various CRC is to obtain easily, comprises the ight soil test (FOBT) of occulting blood, flexible sigmoidoscopy mirror, barium bowel lavage and colonoscopy.The newer method of research is tested as virtual colonoscopy inspection and faeces DNA just energetically.The best approach that detects CRC still remains to be researched and developed.

When comparing with other screenings some in forms, barium bowel lavage (double contrast radiograph barium bowel lavage; DCBE) there is potential benefit, as lower cost (comparing) and lower working time loss with colonoscopy.Some point to the ability that DCBE improves, and come better damage to be positioned the specific anatomical fragment of colon, and this is to use colonoscopy to be difficult to accomplish.This is important situations for operating those of needs.At last, DCBE is safe test, has one of 25,000 expectation percentage of perforation, and the mortality ratio that is caused by one of 25,000 perforation.Should consider the benefit that these are potential at DCBE aspect unfavorable, when comparing with colonoscopy, lack the treatment ability described unfavorable comprising, the uncomfortable property of lower susceptibility and patient is higher.

In 1988 was the average risk filler test of CRC first with the Sigmoidoscope discussion, and become from that time and extensively award the screening technique that allows, or even the preferred test of some associations such as U.S. stomach and intestine research institute (American College of Gastroenterology).

The progress that adenoma changes into the branch subcase understanding that takes place in the CRC process has caused producing the test that suddenlys change among the DNA that flows out in the detection ight soil.DNA in the ight soil is by normal cast and the polyp and the CRC generation of bacterium and mucous membrane of colon.Use a spot of tumour DNA in the molecular biotechnology amplification ight soil.In the last few years, several groups of researchists successfully isolate DNA the ight soil from CRC patient.Based on from the aggregated result that can obtain to study, the faeces DNA test has whole susceptibility for 64% invasion CRC, 95% specificity, for 20% late period adenoma have susceptibility.There is not at present public data about faeces DNA test among total crowd.Faeces DNA test is attractive, because it is a tumour-specific, noninvasive, do not need Enteral formulations or dietary restrictions, and have the single collecting dung quality testing of use and survey the neoplastic potential ability of whole colon length.Present limitation comprises the data that lack to come the self-sizing crowd, needs the test accuracy to measure and needs what or which mark, and the expense of test.

As described, the cost of faeces DNA test at present is high and far away surpasses FOBT's.The tumor marker quantity of restriction test can reduce cost, but this may be under the cost of desensitization.

Recently, several available screening techniques (have for example been commented on by the colorectal cancer advisory group of American Cancer Society (ACS), colonoscopy, the ight soil test (FOBT) of occulting blood, the test of immunochemistry ight soil, capsule endoscope etc.) and the CRC triage techniques of inferring unique recommendation be the test of immunochemistry ight soil, but have strong needs (Levin B etc., CA Cancer J Clin.2003.53 (1): 44-55) for detecting improving one's methods of infantile tumour.

Recognize the needs of the improvement diagnosis of extensive frequent for being used for (annually or 1 year twice) screening, several groups are working to the diagnostic flag in appraisement organization's sample or the blood.

For example, extrasin alpha has been accredited as the potential source biomolecule mark that is used for colon cancer biopsy cell recently, has used new chromatographic technique in conjunction with the SELDI mass spectrum.Another the potential mark that is used for prognosis is a thioredoxin-1, because its expression in tissue sample is very relevant with the stage of colon cancer.Shown that serum thymidine phosphatase can excise the new mark that the hematopoiesis transfer takes place among the CRC patient for prediction.The prognosis that monoclonal antibody also has been used to detect the blood plasma level of CD105 albumen (correctives of angiogenesis) and part TGF β thereof and has proposed CRC patient.Yet the common trait of used test is its low relatively predictive value at present, makes them need replenish other programs, as biological living inspection and/or endoscopy.Consider the defective of existing screening technique, can infer for very the diagnosis of high confidence level need a plurality of protein labelings.

The most popular method of treatment CRC is to remove tumour by operation, or passes through chemotherapy and radiation.Fluorouracil (5-FU) and formyl tetrahydrofolic acid (LV) are usually used in the chemotherapy, and have introduced multiple new reagent recently, comprise Irinotecan (CPT-11) and oxaliplatin, and these cause the raising for the treatment of.The new epoch begin, and wherein chemotherapy is left in treatment, and substitutes to agent with new particular target.Nearest these reagent of use (as anti-angiogenic medicaments, tyrosine kinase inhibitor and epidermal growth factor blocking agent) are finished or ongoing clinical trial provides inspirer preliminary data.

As from can finding out the following prior art of quoting, known protein matter group analysis can identify range protein and with the correlativity of CRC.These protein with regard to they self, can obtain raising or downward modulation, are the indicator of CRC among the patient.One group adjusting pattern also can be used as the indicator of CRC in these protein.These protein can be used as treatment CRC or treat the target of self.

WO05015224 discloses the method for diagnosis colorectal cancer, comprise step: a) provide the fluid sample that obtains from individuality, b) under the condition that the compound that is suitable between described bond and the RLA-O forms, with the particular combination agent of described sample contact 60S acidic ribosomal protein PO (RLA-O), and c) the compound content that forms in (b) is associated with the diagnosis of colorectal cancer.

WO04090550 discloses the method for using colorectal cancer among several protein labeling diagnosis human samples, and mark comprises 60S acidic ribosomal protein PO (RLA-O).The different expression patterns of these marks represent individuality suffer from colorectal cancer and/or the indication colorectal cancer patients disease stage.Use mass spectrometric measurement to identify mark from healthy individual and the various tissues and the blood serum sample that are diagnosed as the colorectal cancer individuality by protein biochip technology.

WO04079368 discloses the specified protein group of methods of expressed protein (comprising HSP90) in the identification of cancer cell.Transfer to identify each mark by on expressing in the colorectal carcinoma sample.Use is reclaimed method of protein from the frozen portions of donor tissue whereby, can draw the normal colonic tissue of selected group and be selected from the patient late period colorectal cancer protein characteristic, select donor tissue based on best tumor histology and cellularity from fresh food frozen tissue bank.

WO00055633 discloses and has identified particular expression feature and the nucleic acid that relates in the colorectal cancer, and this expression characteristic and the purposes of nucleic acid in diagnosis of colorectal carcinoma and prognosis.By protein technique, as mass spectrometric measurement, 2D gel electrophoresis test waits and realizes identifying.This list of references further discloses identifies and uses the candidate agent of adjusting CRC and/or the method for target.

WO02078636 discloses the method and composition that is used to detect and/or treat CRC.More specifically, this list of references discloses the nucleic acid of the colorectal cancer protein of being correlated with and this protein of encoding, and this expression is used for the cell marking of colorectal cancer detection and is used for the molecular targets that colorectal cancer is treated.

WO04021010 discloses the method for diagnosis colon and cancer of the stomach, suffers from various expressions in these disease patient tissues by measurement.Measure expression by the following method, as: (a) detect the mRNA of colon cancer related gene, (b) detect by the related gene coded protein of colon cancer and (c) detect biologically active by the related gene coded protein of colon cancer.

WO04071267 discloses the purposes of NNMT (NNMT) in diagnosis of colorectal carcinoma.In addition, disclose from deriving from the method for individual fecal specimens diagnosis colorectal cancer, by measuring the NNMT in this sample.The measurement of NNMT for example, can be used for the early detection or the diagnosis of colorectal cancer.

WO05015234 discloses the purposes of protein s AHH (S-SAHH) in diagnosis of colorectal carcinoma.In addition, particularly from deriving from the method for individual fluid sample diagnosis colorectal cancer, by measuring the SAHH in this sample.The measurement of SAHH for example, can be used for the early detection or the diagnosis of colorectal cancer.

Before, the success of verified proteome research in identifying such mark is limited.WO9842736 and WO9843091 disclose the particular differences expressed protein mark of identifying by the Proteomic analysis of clinical sample, according to coming the epithelial effort method of enrichment tumour by removing stroma cell and connective tissue pollutant.Yet recently, the protein group comparison normal and tumour colon of the mouse of evaluation does not have statistically-significant difference (Core etc., Electrophoresis 21:1772-81,2000) in protein expression mode.

Following research is also disclosed, about some marks in the various cancerous tissues: Kuniyasu H.Chihara Y.Kondo H.Ohmori H.Ukai R (2003) Amphoterin inductionin prostatic stromal cells by androgen deprivation isassociated with metastatic prostate cancer (amphoterin that exhausts by androgen in the prostate stroma cell induces relevant with metastatic prostate cancer), OncolRep.10 (6): 1863-8; Cappello F.Bellafiore M, Palma A, DavidS.Marciano V, Bartolotta T.Sciume C, (60KDa chaperonin (HSP60) is an overexpression in the colorectal cancer forming process to Modica G.Farina F.ZummoG.Bucchieri F. (2003) 60KDa chaperonin (HSP60) is over-expressedduring colorectal carcinogenesis, European Journal of Histochemistry47 (2): 105-10; Yamamoto S.Tomita Y.Hoshida Y.Sakon M, Kameyama M, Imaoka S.Sekimoto M, Nakamori S.Monden M, AozasaK. (2004) Expression of valosin-containing protein in colorectalcarcinomas as a predictor for disease recurrence and prognosis (containing the prediction agent of the expression of valosin albumen in the colorectal cancer) as palindromia and prognosis, Clinical Cancer Research 10:651-7.

As can be seen, there are the various possibilities of finding the diagnosing tumour mark from the discussion of above prior art.One of the simplest direct method is to use ill tissue itself, but proves that as the present invention prior art can not be identified important CRC labelled protein, as discussed below.

Therefore, fundamental purpose of the present invention is to identify the relevant labelled protein of CRC that did not disclose in the prior art.

Therefore, the invention describes in the table 1 listed target protein, and the method that is used for this application and is used for the CRC treatment is provided as the purposes of CRC mark.

Summary of the invention

The inventor is verified from the metabolic marker of the colon biopsy tissue examination of normal and tumor tissues in conjunction with after 2 dimension gel electrophoresises and the mass spectrum strong instrument that is the protein of identifying that CRC relates in developing.

The inventor has identified the protein relevant with CRC by the existence of the protein of table 1 in the detection of biological sample.Usually, biological sample is selected from urine, ight soil, blood, CSF, saliva, lymph liquid and tissue.Suitably, tissue is the epithelial tissue of colon and/or rectum.Can obviously find out from the viewpoint of diagnosis, urine, ight soil, saliva or blood are preferred.

The Proteomic analysis that is applied to such biomaterial has shown protein level has been caused the mechanism of CRC development and the understanding of identifying related protein in CRC diagnosis or the treatment.

Opposite with prior art cited above, the inventor has used fresh tissue sample,, does not have the sample of freezing or other storage that is, and storage will cause the degraded of some unique protein labelings.Resection organization's sample is transferred to them in the cell culture medium immediately as early as possible, and with radioactive isotope (for example, 16[ ¹⁴C]-amino acid or [ ³⁵S]-potpourri of methionine) metabolic marker, this guarantees that the statement of Proteomic analysis that can be by prior art comes any protein of expressing in the certification mark interval procedure, even be a few minutes at interval.

As discussed in detail below, target protein of the present invention has special-purpose, particularly as diagnosis and the prognostic markers of cancer (particularly CRC).

Known mark is the same with using, and for example, they can be used for the existence of auxiliary diagnosis progression of disease early-stage cancer and predict the possibility of successful result clinically, particularly about susceptibility or the resistance of specific patient tumors to chemotherapeutics or chemotherapeutics composition.In addition, these targets can be used for the treatment intervention of colorectal cancer or other cancers, for example, target tumor cell and reduce toxicity in the health tissues specifically, with evaluate candidate treatment compound be provided regulated from colon the method for the bioactive ability of the cancer cell of rectum and its hetero-organization.

Therefore, the present invention relates to the diagnosis and the treatment of cancer, particularly distinguish tumour cell and normal cell, with by utilizing in the tumour cell different expression of these antigens to aim at treatment based on the overexpression of particular tumor antigens.

The detection (referring to table 1) of one or more protein (" labelled protein " or " target protein ") of overexpression in the tumour cell The present invention be more particularly directed to compare with the expression in the normal cell.

The present invention further provides the method for aiming treatment of cancer treatment, by guiding the protein of these overexpressions of treatment antagonism.The method that realizes this aiming can comprise, but be not limited to: (i) medicine and the target protein of specific recognition target protein molecular structure or combining of adaptive subdivision, (ii) use protein, polypeptide, expression vector or dna vaccination construct, by immunity host immune system is exposed to target protein or its fragment, so that guide host immune system to resist the wherein tumour cell of target protein overexpression, (iii) regulate the biologically active of target protein by the micromolecule part, (iv) utilize the biologically active of target protein to activate prodrug, (v) pass through as the antisense gene montage, use siRNA molecule, or the regulating and controlling sequence in the aiming coding target protein gene or regulate the expression of target protein in the cell in conjunction with the such method of the adjusting albumen of these sequences, (vi) other compositions of target protein and cell, for example, with the physical interaction particular adjustments of micromolecule part or immunoglobulin (Ig), so that performance treatment benefit.Therefore, the difference expression that the present invention is based on target protein provides various new methods, is used for cancer, comprises the diagnosis of CRC, prognosis and treatment.When considering following detailed Description Of The Invention, these and other many aspects of the present invention and advantage are that those skilled in the art are conspicuous.

Described in claim, the present invention is based on the existence of mensuration from least one labelled protein in the described mammiferous biological sample, does not exist or expression, and wherein labelled protein is selected from down group:

The protein that limits in the table 1; With

Be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology.Described modification can include, but are not limited to: N ' or C ' block, splice variant, acidylate, acetylation; adenylylation, ADP-ribosylation iodate, biotinylation, carbamylization, carboxymethylation; desamidization, esterification, farnesylation, formylation, glutathione; glycosylation, hydroxylation, fatization methylates, the nutmeg acidylate; oxidation, palmitoylation, phosphorylated, isoprenylation, sialylated (sialation); stearic acidylate, sulfonation, sulfuration, vitamin C-independently modify, and selenoprotein with K-.

Yet, because prior art discloses the purposes that HSP90 and 60S acidic ribosomal protein are used to detect CRC, theme of the present invention is based on the mensuration of the existence of another labelled protein at least that is selected from this group, when described at least a labelled protein was HSP90 and/or 60S acidic ribosomal protein, this another labelled protein was not HSP90 or 60S acidic ribosomal protein.This also is applicable to the protein from table 1 group A, and known its obtains/reduce in the CRC tissue.

Detailed Description Of The Invention

In first aspect, the present invention relates to the protein of identifying by 2D-gel electrophoresis and mass spectrum.

Relate in one aspect to the purposes of the protein of table 1 again, and the purposes that relates to protein as CRC mark or indicator, the existence of this protein, do not exist or popularity degree (prevalence) uncorrelated with CRC before.Think the change of protein expression and protein expression mode for diagnosis, prognosis and treatment are used and target is important mark.

Protein of the present invention can a) relate to energy transduction and oxidation-reduction potential and glycolytic ferment; B) relate to purine/pyrimidine and synthesize, DNA/RNA is synthetic, RNA processing, amino acid metabolism and protein synthesis; C) as chaperone; D) relate to differentiation, apoptosis, regulation and control and signal transduction; E) relate to cytophylaxis; F) relate to eucaryotic cell structure; G) relate to hormone/neurotransmitter metabolism; H) or have other functions.

First aspect of the present invention relates to labelled protein.Labelled protein is selected from down group: a) protein of table 1; And b) have and a) in the protein of protein at least 80% sequence homology.

In addition, labelled protein can be selected from: a) with respect to impinging upon one or more protein that exist with remarkable lower or remarkable high level from the protein polyacrylamide gel of described biological sample; B) non-existent one or more protein on the polyacrylamide gel of the protein of contrast from existing on the polyacrylamide gel of the protein of described biological sample; And c) do not exist on the polyacrylamide gel from the protein of described biological sample and one or more protein of existing on the polyacrylamide gel of the protein that contrasts.

The meaning of term " contrast " is conspicuous for the technician in the proteomics field.Preferably, contrast comes in the IOD% value of the spot (having identical position) of protein gel, protein from be derived from do not tend to or suffer from CRC the people sample or from the healthy fragment of the colon of suffering from CRC patient.Term " labelled protein " meaning is to comprise above-mentioned protein, and from the similar protein matter of other mammalian species that are used for the CRC experiment.People's form of protein is to make us interested especially.For example, the rat form of raiser albumen can be as the mark of CRC in the rat experiment model.Therefore, term " labelled protein " comprises the mammal form of people's albumen of identifying in the table 1.

Another aspect of the present invention relates to diagnosis or measures for example tendentious method of people CRC of mammal, and this method comprises the existence of measuring at least a labelled protein in the described mammiferous biological sample, does not exist or expression.Biological sample is selected from urine, ight soil, and blood, saliva, lymph liquid and tissue, fresh blood, ight soil, urine, saliva or colonic epithelium tissue are preferred.

Preferable methods comprises the raising expression (labelled protein of rise) of setting up at least a labelled protein at present, and labelled protein is selected from down group: a) limit in the table 1, and be labeled as the protein of rise; And b) is the trim of a) middle protein or the protein of derivant, makes to have a) at least 80% sequence homology of middle protein, the invention still further relates to the method for the reduction expression of setting up at least a downward modulation labelled protein certainly.

In another embodiment, the present invention relates to measure the tendentious method of people CRC, this method comprises: the raising of a) measuring at least a labelled protein in the biological sample that is derived from the people is expressed, described labelled protein is selected from down group: i) the upregulated protein matter that limits in the table 1, or ii) be i) in the trim of protein or the protein of derivant, make and to have i) at least 80% sequence homology of protein, b) reduction of at least a labelled protein is expressed in mensuration people's the biological sample, described labelled protein is selected from down group: i) the down-regulation protein matter that limits in the table 1, or ii) be i) in the trim of protein or the protein of derivant, make and to have i) at least 80% sequence homology of protein, or c) a) and b) in the combination determined.

Therefore, protein is the useful indicator of determining can be used as the CRC neurological susceptibility that raises or reduce.The last pattern that is in harmonious proportion downward modulation also can be used as indicator.That is to say and set up, and the expression pattern of a histone matter is used as indicator more than a kind of protein expression level.

In suitable embodiment, at least a labelled protein is selected from respect to impinging upon one or more protein that exist with remarkable lower or remarkable high level from the protein polyacrylamide gel of described biological sample, non-existent one or more protein on the polyacrylamide gel of the protein of contrast from existing on the polyacrylamide gel of the protein of described biological sample do not exist on the polyacrylamide from the protein of described biological sample and one or more protein of existing on the polyacrylamide of the protein that contrasts.

In another embodiment, the present invention relates to treat for example method of people CRC of mammal, or the method for prevention or delay CRC outbreak, comprise the expression that changes at least one labelled protein.

This method preferably includes and can change compound administration that expression returns towards the control (control) of one or more protein in the people, and protein is selected from: a) protein that limits in the table 1; And b) is the trim of table 1 protein or the protein of derivant, feasible at least 80% sequence homology with table 1 protein; C) have a) and/or b) in the protein of protein identical function; D) coding a), b) or c) in the nucleotide sequence of protein; E) a), b) or c) antibody of protein; F) can be in conjunction with a), b) or c) nucleic acid fragment of protein; And g) can be in conjunction with a), b) or c) compound of protein.

In addition, the present invention relates to the purposes that this compound is used to make treatment or prevention CRC medicine.Term " colorectal cancer " or its abbreviation " CRC " comprise the disease relevant with colorectal cancer.

About the method for treatment CRC, can the single albumen of target, or can be used for the treatment of by target one histone.Can change these by the expression of targeting proteins, or can interferencing protein self, so that change their activity.Therefore, an interesting embodiment of treatment people CRC method comprises the expression that changes labelled protein.

In another embodiment, the present invention relates to measure reagent has the possibility of result of treatment in the CRC treatment method, comprise that measuring test model is exposed to before the described reagent and relative expression's level of one or more labelled proteins afterwards.

In addition, the present invention relates to measure the method for the effect of compound in the CRC treatment, comprise the protein expression level of measuring one or more labelled proteins.

Again in the embodiment, the present invention relates to measure the method for the effect level of compound used therefor in the CRC treatment, comprise and measure the expression that test model is exposed to one or more labelled proteins before or after the described reagent.

In addition, the present invention relates to measure ill or easy ill the mammal for example character of people CRC or the method for inducement, comprise and set up at least a expression that relates to the labelled protein of model.

Another embodiment of the invention relates to the nucleic acid fragment of the nucleotide sequence that comprises coded markings albumen, or relate to this nucleic acid fragment or its part or with the nucleic acid fragment of its complementary strand hybridization.The invention still further relates to above-mentioned nucleic acid fragment and be used for the purposes that certification mark albumen exists.

Again in the embodiment, the present invention relates to can incorporation of markings albumen antibody.Such antibody can be polyclonal antibody or monoclonal antibody.The invention still further relates to this antibody and be used for the purposes that certification mark albumen exists.

Again in the embodiment, the present invention relates to be used to diagnose mammal for example people's CRC or CRC genetic predisposition's testing cassete, comprising: a) specificity is in conjunction with the combination tool of at least a labelled protein; Perhaps this combination tool is the antibody of at least a described labelled protein, can be in conjunction with the nucleic acid fragment of at least a described labelled protein, or can be in conjunction with the compound of at least a described labelled protein; B) instrument that detects combination tool and at least a labelled protein or combine with at least a nucleic acid fragment, if existence combination, perhaps for detecting instrument in conjunction with level, with c) be used for related instrument, combining of related and described combination tool, if the existence combination is perhaps related in conjunction with level, whether represent that individual mammal has the possibility of significantly higher trouble CRC or the genetic predisposition of trouble CRC.

In another embodiment, the present invention relates to measure the method for material effect, this method comprises uses mammal, people for example, it has used method of the present invention to be established as to have the possibility of suffering from CRC or the high individuality of genetic predisposition of suffering from CRC, and this method comprises the effect that material is delivered medicine to individuality and measure this material.

The inventor expects that measuring method ill or that be easy to ill people's the character of CRC or inducement comprises and set up the expression of table 1 protein about model that this model is used for understanding disease and potential treatment.

In the test of compound, be useful and help to improve described treatment for the therapeutic activity of understanding described reagent about the activity of reagent or the knowledge of target.

The inventor's research and development have produced the method for measuring the effect of compound in the CRC treatment, comprise the expression of measuring one or more labelled proteins, and relate to the expression of compound used therefor in the mensuration CRC treatment or the method for activity level, comprise that measuring test model is exposed to before the described reagent and the expression of one or more labelled proteins afterwards.

The very important embodiment of the present invention relates to pharmaceutical composition, and it comprises: a) can regulate the material that nucleic acid fragment is expressed, at least a portion of this nucleic acid fragment coded markings albumen; B) labelled protein; C) derivant of labelled protein, homologue or analogies; D) antibody of labelled protein; E) nucleic acid fragment that can incorporation of markings albumen; And/or f) compound that can incorporation of markings albumen.

Pharmaceutical composition can be used as medicine, as is used for the treatment of or prevents CRC.

It should be noted that expection will make analysis become the more reliable indication of CRC relevant disease more than a kind of detection of combination in any of mark.Therefore, comprise the existence of measuring two kinds of marker combination, activity, the diagnosis of concentration and/or expression or to measure the tendentious method of CRC be preferred, strong preferred three kinds or multiple mark (for example, at least 4,5,6 or 7 kind of mark).Expection can improve the specificity and the sensitivity of diagnosis more than a kind of detection of labelled protein, therefore improves the value of diagnostic test.

Also similarly advised using more than a kind of (for example, at least 2,3,4,5,6 or 7 kind of compound) according to compound of the present invention (for example, be selected from more than a kind of compound: according to polypeptide of the present invention, nucleic acid fragment or antibody) treatment, the compound of described combination can influence the level more than a kind of labelled protein, will make treatment of diseases more effective.In this, may even expect that the treatment of only using a kind of compound will influence the expression more than a kind of labelled protein.

Term among the present invention " polypeptide " will have its common meaning.Be the amino acid chain of any length, comprise full-length proteins, oligopeptides, small peptide and fragment thereof wherein connect amino acid residue by the covalency peptide bond.Term " polypeptide ", " peptide ", " amino acid sequence " and " protein " can be used alternatingly.

Can pass through phosphorylation, methylate, sulphation (sulphylated), glycosylation or by adding any type of lipid or fatty acid, ubiquitin or any other side group or the modification by containing other amino acid or any other form (wherein exist kind more than 200 known) are at modifying protein chemically or on the biological chemistry.These modifications occur in the specific site of protein, and the specific modification in a site can have different effects with the identical modification of different loci on the same protein.They are reversible in cell, and for example they are used for opening or closing enzyme in cell, thus protein can exist with various forms-each has the related activity that is used for each protein function.In addition, can spilit-polypeptides, for example, handle its N-or C-and bring in and remove signal peptide, perhaps can remove internal sequence by montage mRNA, during translation, it will form the protein that changes.Can in Protein Data Bank such as EXPASY, find examples many in these, and exist the instrument of growing scope to predict these modifications and function (referring to http://www.expasy.ch) thereof.Because estimate that each protein among the mankind is on average modified 10-20 time, the most of protein that is expected at this evaluation obtains modifying in this or the sort of mode.

Therefore, each polypeptide can characterize by specific amino acid and be encoded by specific nucleotide sequence.Comprise analog and the variant that produces by reorganization or synthetic method with understanding such sequence, wherein such peptide sequence is by displacement, insert, add or the deletion recombinant polypeptide in one or more amino acid residues and obtain modifying, and remain immunogenic in described herein any biological test.

Displacement is preferably " guarding ".Conservative substitution is well known to a person skilled in the art.Preferably, consider to belong to phase amino acid (nonpolar (G, A, P, I, L and V), polarity-uncharged (C, S, T, M, N and Q), polarity charged (D, E, K and R) and aromatic (H, F, W and M) on the same group.In these groups, amino acid can the phase double replacement, and other displacements also are fine certainly.

Each polypeptide is by specific nucleic acid sequence encoding.Comprise its analog and variant with understanding such sequence, wherein such nucleotide sequence inserts by displacement, adds or delete one or more nucleic acid residues (to comprise and insert one or more intrones (little or big) and obtain modifying.Replace the silence displacement during preferably codon uses, this can not cause any change of amino acid sequence, improves protein expression but can introduce.

In this article, term " the pure basically polypeptide " meaning be contain at the most the polypeptide formulations of 25% weight and its natural other peptide materials that link to each other (other peptide materials of preferred lower number percent, for example, at the most 20%, at the most 15%, at the most 10%, at the most 5% and at the most 1%).Preferred pure basically polypeptide is at least 96% pure, that is, polypeptide constitutes at least 96% weight of the total peptide material that exists in the preparation, and preferred higher number percent, as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5% and at least 99.75%.Especially the preferred polypeptide fragment is " a pure basically form ", that is, polypeptide fragment essentially no any other with its natural protein that links to each other, that is, do not have any other from mammiferous protein.This can realize by preparing polypeptide of the present invention, by the recombination method in the host cell well known by persons skilled in the art, or by solid phase or the synthetic synthetic polypeptide fragment of known method of liquid phase peptide, for example, by the described method (Merrifield of Merrifield, R.B.Fed.Proc.Am.Soc.Ex.Biol.21:412,1962 and J.Am.Chem.Soc.85:2149/1963) or its improve one's methods, or by from running gel, retrieving to prepare polypeptide of the present invention.

Term " protein " also comprises derivant, analog and the analogies of aforementioned polypeptides.Such derivant, analog preferably has identical activity with the polypeptide that produces it with analogies, for example, the enzymatic activity of identical type.Derivant, analog or analogies can have the activity than parent's polypeptide reduced levels, identical level, or preferred, higher activity level.

Term " at least a " (for example, at least a compound or at least a labelled protein) comprises integer 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65 and 66.Being to be understood that and can using single marking albumen, may be favourable but use in the method for the invention more than a kind of labelled protein, that is to say the expression of foundation more than a kind of albumen, and the expression pattern of a histone is used as indication.Obviously along with the increase of number in the group, improved the reliability of identifying.

" peptide mimics (peptidomimetic) " is the biologically active of simulating peptide but no longer is the molecule of peptide on chemical property.Strict definition is: peptide mimics is the molecule that no longer contains any peptide bond (that is the amido link between the amino acid).Yet it not exclusively is the molecule of peptide that the term peptide mimics sometimes is used on the descriptive nature, as false peptide (pseudo-peptide), and half peptide (semi-pept ide) and class peptide (peptoid).No matter be non-peptide wholly or in part, peptide mimics according to the present invention provides the spatial disposition of reactive chemical part, its to peptide mimics based on peptide in the three-dimensional arrangement of reactive group extremely similar.As the result of this similar avtive spot geometry arrangement, peptide mimics has effect to biosystem, and its biologically active to peptide is similar.The present invention includes the peptide mimics composition, it is the analog of simulation according to the activity of biologically active peptide of the present invention, and promptly peptide mimics can be used for treating the CRC relevant disease.Preferred peptide mimics of the present invention is similar basically to above-mentioned listed peptide or avtive spot on three-dimensional structure and biologically active.

Perhaps, analogies can be " anti-analogies (antimimetic) ".In other words, be suitable for and the molecule of blocking protein avtive spot, or in conjunction with binding site or with the molecule in the site of other biological interaction of molecules, make the function of interferencing protein.Most drug is such at present.The present invention includes can with the interactional this anti-analogies of polypeptide of the present invention.

Use the analogies of given peptide to have remarkable advantages, because peptide presents two disadvantageous features usually: (1) biological utilisation rate variance than peptide itself; (2) duration of effect is short.Peptide mimics has given significantly to walk around the path of these two major obstacles because the molecule of paying close attention to is enough little, make be Orally active and have a long acting duration.Also there are the remarkable cost savings relevant and patient's compliance of raising,, can be taken orally because they are compared with the non-enteron aisle or the mucosal of peptide with peptide mimics.In addition, the production of peptide mimics is than peptide considerably cheaper.At last, the stability relevant with peptide stores and immunoreactive problem did not run in peptide mimics.

Therefore, above-mentioned peptide can be used to research and develop so little chemical compound that therefore similar biologically active also has similar therapeutic efficiency that has.The technology of research and development peptide mimics is conventional.Therefore, can substitute peptide bond, make peptide mimics adopt the structure similar, and therefore have the biologically active similar to initial peptide to initial peptide by non-peptide bond.In addition, also can substitute amino acid whose chemical group with the chemical group of other analog structures modifies.By spectroscopy, the tertiary structure that initial peptide is measured in crystallography and/or Computer-aided Molecular modeling is assisted the research and development of peptide mimics.These technology help research and development than more efficient and/or higher bioavailability of initial peptide and/or more stable new compositions (Dean (1994), BioEssays, 16:683-687; Cohen and Shatzmiller (1993), J.Mol.Graph.11:166-173; Wiley and Rich (1993), Med.Res.Rev., 13:327-384; Moore (1994), TrendsPharmacol.Sci., 15:124-129; Hruby (1993), Biopolymers, 33:1073-1082; Bugg etc. (1993), Sci.Am., 269:92-98 all is incorporated herein by reference document at this).In case identify a kind of potential peptide simulated compound, can be with it synthetic and use diagnostic test described herein or suitable diseases inhibitors to test and evaluate its activity (referring to (1983) such as Finlay, Cell, 57:1083-1093 and Fujiwara etc. (1993), Cancer Res., 53:4129-4133 is hereby incorporated by document).

Therefore, utilize said method, the invention provides with aforementioned polypeptides and compare, present the compound of the therapeutic activity of raising.The present invention includes the peptide simulated compound that obtains by said method, it has the biologically active of above-mentioned peptide and similar three-dimensional structure.Those skilled in the art will know that peptide mimics can originate from any of above-mentioned modified peptides or originate from the peptide that has above a kind of above-mentioned modification.In addition, except the purposes as the treatment compound, peptide mimics of the present invention can be further used for researching and developing more effective non-peptide compound with clear.

Term " nucleic acid fragment " and " nucleotide sequence " etc. are interpreted as any nucleic acid molecules, comprise DNA, RNA, LNA (nucleic acid molecules of locking), PNA, RNA, dsRNA and RNA-DNA heterozygote.The nucleic acid molecules that also comprises the nucleosides that contains the non-natural generation.This term comprises the nucleic acid molecules of any length, for example, 10 to 10000 nucleotide, this depends on purposes.When nucleic acid molecules as DNA treatment in medicine or be used for when producing method according to polypeptide of the present invention, preferred use is encoding to the molecule of small part polypeptide, has about 10 length to about 1000 nucleotide, and this molecule is randomly inserted in the carrier.When nucleic acid molecules as probe, primer or when being used for antisense therapy preferably uses the molecule of length as 10-100 nucleotide.According to the present invention, can use the molecule of other length, for example, have at least 12,15,21,24,27,30,33,36,39,42,50,60,70,80,90,100,200,300,400,500 or 1000 nucleotide (or nucleotide derivative), or have at the most 10000,5000,4000,3000,2000,1000,700,500,400,300,200,100,50, the molecule of 40,30 or 20 nucleotide (or nucleotide derivative).Be to be understood that these numerals can produce various scopes by independent assortment.

Term when using in conjunction with hybridization conditions " rigorous " is identical with the definition in this area, that is, hybridize under 15-20 ℃ the temperature that is no more than under the nucleic acid fragment fusing point (Tm), with reference to Sambrook etc., Molecular Cloning; A Laboratory manual (molecular cloning; Laboratory manual), Cold Spring Harbor Laboratories, NY, 1989, p11.4511.49.Preferably, condition is " highly rigorous ", and promptly nucleic acid fragment fusing point (Tm) is 5-10 ℃ down.In the present invention, preferred hybridization conditions is rigorous.In whole instructions, unless requirement in addition in the literary composition, term " comprises " or its distortion as " containing " or " comprising ", will be interpreted as that expression comprises described key element or whole or one group of key element or integral body, but do not get rid of any other key element or whole or one group of key element or integral body.

Term " sequence homogeneity " (or " sequence homology ") refers between the amino acid sequence of two equal lengths or the quantitative measurment of homology degree between the nucleotide sequence of two equal lengths.If two sequences to be compared are not isometric, they must be arranged in most probable coupling with insertion that has breach or the mode of cutting off at the protein sequence end.Can be according to (Nref-Ndif) * 100/Nref sequence of calculation homogeneity, the sum of different residues in two sequences when wherein Ndif is for comparison, wherein Nref is the residue number of one of sequence.Therefore, has 75% sequence homogeneity (Ndif=2 and Nref=8) between dna sequence dna AGTCAGTC and the sequence MTCAATC.Breach is counted different specific residues, promptly have 75% sequence homogeneity (Nd f=2 and Nref=8) between dna sequence dna AGTG C and the dna sequence dna AGTCAGTC.

Perhaps, sequence homogeneity can be calculated by blast program, as blast program (Person W.R and D.J.Lipman (1988) PNAS USA 85:2444-2448) ( Www. Ncbi.nlm.nih.gov/BLAST/), or at ExPasy website (op.sit.) acquisition blast program.In one aspect of the invention, can compare with the sequence alignment method ClustalW that has default parameter, as Thompson J. etc., described in the Necleic Acids Res 199422:4673-4680, http://www2.ebi.ac.uk/Clustalw.Perhaps, use is used for the standard punishment of DNA: GAP weight 5.00, length weight 0.300 from the homology degree between two nucleotide sequences of GAP mensuration of the version 8 of GCG bag, matrix described in Gribskov and the Burgess, Nucl.Acids Res.14 (16); 6745-6763 (1986), use is from GCG bag (Genetics Computer Group, 575ScienceDrive, Madison, Wis.53711, the GAP of version 8 USA) measures the homology degree between two amino acid sequences, use is used for the standard punishment of protein: GAP weight 3.00, length weight 0.100, the matrix described in Gribskov and the Burgess, Nucl.Acids Res.14 (16); 6745-6763 (1986).

The preferred minimum percent of sequence homology is at least 70%, as at least 80%, and at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5%.

The invention still further relates to polypeptide of the present invention or nucleic acid purposes as the treatment vaccine, as Lowry, D.B. etc., 1999, described in the illustrational document of Nature 400:269-71.

The monoclonal or the polyclonal antibody that react with polypeptid specificity of the present invention in immunoassay, or the specificity binding fragment of described antibody also are parts of the present invention.

Can produce antibody by method known to those skilled in the art.Can in mammal, produce polyclonal antibody, for example, inject according to polypeptide of the present invention by one or many, and if desired, can the one or many injection adjuvant.Can be by Kohler and Milstein at Nature, the hybridoma method of describing first among the 256:495 (1975) or as U.S. Patent No. 4,816, the recombinant DNA method production described in 567 is according to monoclonal antibody of the present invention.For example, also can use McCafferty etc. at Nature, the technology of describing among the 348:552-554 (1990) is isolated monoclonal antibody from phage library.Described the method for producing antibody in the document, for example the U.S. 6,136, and 958.

In diagnosis, in treatment or the test, can use antibody of the present invention, nucleic acid fragment and/or polypeptide separately, also can be used as the composition in the composition.Such composition is known to those skilled in the art, and comprises antibody wherein of the present invention, nucleic acid fragment or polypeptide combination, and preferably covalently is in conjunction with the composition of at least a other molecule such as mark (for example, radioactivity or fluorescein) or carrier molecule.

The invention further relates to the method for using above-claimed cpd therapeutic and/or preventative processing CRC relevant disease patient.

Method of the present invention comprises step: a) one or more compounds of the present invention are mixed in the suitable pharmaceutical carrier; And b) will mix in the carrier the treatment effective dose or the prevention effective dose compound administration in the patient.

Term " suitable pharmaceutical carrier " refers to any carrier known in the drug world, is used for compound administration in the patient.Can use any suitable pharmaceutical carrier according to the present invention, the short of consistency problem that causes.

Can be by non-enteron aisle injection, as intravenous, intramuscular or intra-arterial are realized to patient's administration effective dose.These compounds also can oral or percutaneous dosing, perhaps by any other method well known by persons skilled in the art, as by inhalator or nasal atomizer.Oral is preferred at present.

As used in this, term " treatment effective dose " refers to the content of one or more compounds of the present invention of therapeutic treatment needs of patients.Such processing is suitable for being diagnosed as the patient who suffers from the CRC relevant disease.Similarly, term " prevention effective dose " refers to the content of one or more compounds of the present invention of preventative processing needs of patients.Such processing is suitable for following patient, for example, does not also set up any clinical symptoms of CRC relevant disease.Determining that it will be favourable beginning preventative processing as early as possible after the patient is in the risk that produces the CRC relevant disease, for example, measure the tendentiousness of CRC by the mark with change level.

It will be appreciated by those skilled in the art that, the dosage of given compound, administration path and treatment duration not only depend on the type and the effect in this disease of treatment thereof of compound, but also depend on the individuality of being treated, consider such factor, as weight in patients, be used for the treatment of patient's other methods of treatment and patient's situation, clinical response and tolerance.Those skilled in the art can determine dosage, administration and duration by evaluating these and other correlative factor.

The invention provides people CRC-mediation albumen, that is, be accredited as and relate in the CRC production process or affected protein.CRC-is mediated albumen be characterized by such albumen, it be expressed in the CRC production process with respect to not existing the expression of CRC under producing to obtain change.The present invention has identified CRC-mediation albumen from the 2-Wei Ningjiaoshuojuku of people's colon/rectum biopsy cut sections for microscopic examination and epithelial cell albumen.CRC-mediation albumen comprises protectiveness CRC-mediation albumen and harmful CRC-mediation albumen.

The invention provides people CRC-mediation albumen by using 2-dimension gel to identify, 2-dimension gel can comparative control and the cancer epithelial cell identify which protein replys, identify the protein that in cell response, works.Connect and analyze the functional group and the regulating action (for example, by tyrosine phosphorylation or other posttranslational modifications) thereof that can be used for limiting protein.

The invention provides the protectiveness CRC-mediation albumen (" protective protein ") of purifying basically, it is characterized by to protect to be in and produce the generation that the patient in the disease risks resists CRC, or alleviate or alleviate the patient's who suffers from CRC CRC symptom.Protective protein of the present invention can directly be used for protection antagonism CRC, maybe can by induce or improve the synthetic of second kind of protective protein by reduce or suppress harmful albumen synthesize to come indirectly-acting.The present invention comprises that further the full length amino acid sequence with people CRC-mediation albumen has at least 80%, preferably at least 90%, more preferably at least 95% and the amino acid sequence of at least 98% homogeneity most preferably.For example, measure number percent homology or homogeneity by comparative sequences information, use the GAP computer program, version 6.0 can obtain from University of Wisconsin Genetics Computer Group (UWGCG).The GAP program is used the comparison method of Needleman and Wunsch (1970), J.Mol.Biol.48:443, according to Smith and Waterman (1981) revision, Adv.Appl.Math.2:482.In brief, the GAP program is defined as similarity the number of similar comparison symbol (that is, nucleotide or amino acid) divided by the short total number of symbols of two sequences.The preferred default parameter that is used for the GAP program comprises: (1) monobasic relatively matrix (contain for identical be 1 with for different be 0 value) and the weight of Gribskov and Burgess (1986) Nucl.Acids Res.14:6745 matrix relatively, described as Schwartz and Dayhoff (editor), (1979) Atlas Of ProteinSequence And Structure (protein sequence and structure atlas), NationalBiomedical Research Foundation, pp.353-358; (2) 0.10 other punishment of each symbol in the punishment of each breach 3.0 and each breach; (3) for the not punishment of terminal breach.

The present invention further comprises the polynucleotide sequence of code book invention CRC-mediation albumen, comprises DNA, cDNA, PNA and RNA sequence.It is also understood that all polynucleotide that also comprise all or part of CRC-mediation of coding albumen at this, as long as their codings have the active polypeptide of CRC-mediation.Such polynucleotide comprise natural generation, polynucleotide synthetic and that have a mind to manipulation.

For example, such polynucleotide can be accepted orthomutation.Polynucleotide sequence of the present invention also comprises antisense sequences.Antisense sequences comprises with the synthetic sequence of the oligonucleotides of modifying.Polynucleotide of the present invention comprise as the result of genetic code and the sequence of degeneracy.Have 20 natural amino acids, wherein major part is by more than one codon appointment.Therefore, the present invention includes all degenerate core nucleotide sequences, as long as CRC-mediation amino acid sequence of polypeptide is by unaltered nucleotide sequence coded on the function.

Harmful CRC-mediation albumen (" harmful albumen ") is characterised in that improving the patient produces CRC or improve the risk that the patient produces CRC.

Two-dimensional gel electrophoresis (2-DGE) is instrument (for example, Andersen etc., (1995) Diabetes 44:400-407 of especially effectively isolated protein potpourri; John NE etc., Diabetes (2000); 49:1819-29.Christensen etc., Autoimmunity. (2000); 32:1-15 and Mose Larsen etc., Diabetes. (2001); 50:1056-63).The cell protein extract is placed on the gel, at first, go out single protein by size separation then by electric charge.The result is the characteristic pattern of many as 1,000 to 5,000 point, and each constitutes single protein usually.By increasing the gel size, by using Radiolabelling method, silver dyes raising sensitivity, and gel thicknesses is reduced to 1.5mm and littler, improves resolution.Can be by on the first dimension gel that covers narrow pH range (for example, 1.5,1pH unit or lower), running the further raising that glue obtains resolution.Described in following examples, can identify the single protein that reclaims from the 2D gel by mass spectrum, obtain trypsase clastotype and the accurate molecular weight of each peptide.Then these observed values are used for searching in DNA and Protein Data Bank, measure the coupling that whether exists with the protein of identifying before.Can be from known protein matter mensuration homogeneity or from inferring with the high homology of known protein matter.

The 2D electrophoresis is used for separating and identifies in the CRC production process, present when changing synthetic protein spots, the protein spots of identifying from the gel excision, and produce peptide with trypsinization.From the gel recovering peptide and accept mass spectrum (substance assistant laser desorpted/MALDI-MS-MALDI), and with the computerize MS-characteristic pattern of resulting MS-characteristic pattern, and analyze facing to suitable sequence information facing to all sequences that finds in public's sequence library.If obtain and any coupling of clone's sequence before, collect information about corresponding gene and coded protein.When the CRC-mediation albumen of identifying is not complementary with the protein of cloning before, can protein micrometering preface be obtained partial amino-acid series information by methods known in the art.Can also protein (when obtaining with q.s) part be checked order by nanometer esi-msn for example, wherein in gas phase with specific fragments of peptidesization, and the molecular weight of fragment is used for producing partial amino-acid series.In case can obtain partial sequence information, those except mentioning before can also be searched in cDNA or EST (the sequence mark thing of expression) database so.

Based on the result who obtains from database search or amino acid sequencing, make up specific with primer degeneracy and be used for screening the partial sequence that people's colon and/or rectum epithelium library maybe will be used for cloning corresponding cDNA by the first chain cDNA of PCR.

Then the sequence that obtains being used for obtaining the full length coding region territory, by 5 '-race PCR or by the conventional hybridization triage techniques, then is Recombinant Protein Expression (Karisen etc., (1991) Proc.Natl.Acad.Sci.USA 88:8337-8341; Karlsen etc., (1994) exist: Insulin secretion and pancreatic bata-cell research (insulin secretion and the research of pancreas beta cell), Flatt, P.R. etc., Smith-Gordon, USA; The 64th chapter, pp.1-9; Karlsen etc., (1995) Diabetes 44:757-758).

Can come separation of C RC-mediation albumen with variety of way well known by persons skilled in the art, comprise from the biomaterial purifying, and from recombinant dna expression (referring to above).The conventional method step comprises extraction, precipitation, chromatogram, affinity chromatography and electrophoresis.For example, can express the cell of CRC-mediation albumen by centrifugal collection, or use suitable reducing, cracking, and by column chromatography, for example, at DEAE-cellulose, cellulose phosphate, polyribocytidylic acid-agarose on the hydroxylapatite, or by electrophoresis or immunoprecipitation, comes isolated protein.Perhaps, can come separation of C RC-mediation albumen by the immunoprecipitation that uses specific antibody.

Method of testing provided by the invention can be used for screening can be influenced CRC-mediation protein expression and therefore influence the compound that people CRC produces.The model that a kind of screening can influence the medicine of one or more CRC-mediation protein expressions is to give cells in culture with the compound (comprising antisense oligonucleotides) that suspection has a beneficial effect.Useful cell is the cell that produces of colon or rectum particularly.Can measure the effect of test compounds by the 2D gel electrophoresis then to protein expression.

Another screening model is to use and is in method in the mammiferous body that produces the CRC risk.In brief, (for example, CRC tendentiousness rat or mouse) mammal is exposed to test compounds, and measures the effect that test compounds produces CRC that is exposed to will to have the CRC risk of raising.

Can be in producing the whole process in stage the generation of monitoring CRC, expression and time under the expression by measuring one or more CRC-mediation albumen and the time of comparison seizure of disease and disease do not produce.The expression of measuring one or more CRC-mediation albumen comprises CRC-mediation albumen self, the modified outcome after the translation, and/or CRC-mediation protein degradation product.In one embodiment, measure the activation of CRC-mediation albumen by the level of measuring CRC-mediation protein expression in the specimen.Suitable specimen comprises body fluid, as blood, and urine, ight soil is tied rectal tissue or cerebrospinal fluid, or is derived from these fluid, as blood plasma or serum.In specific embodiment, measure the level of protein expression in the specimen by western blot analysis., be transferred on the film the protein fractionation that exists in the sample by gel electrophoresis, and survey with the protein-specific antibody of mark.

In another specific embodiment, measure the level of CRC-mediation protein expression by rna blot analysis.Separate polyadenylation [many (A)+] mRNA from specimen., and be transferred on the film the mRNA classification by electrophoresis.CDNA with mark comes detection membrane.In another embodiment, the quantitative PCR of the mRNA by being applied to expression is measured protein expression.

Again in the one side, the invention provides the method that evaluation can suppress or reduce the compound of endogenous harmful protein expression, and the method that prevents and/or treats CRC by the compound that can suppress or reduce endogenous harmful protein expression of drug treatment effective dose.

CRC-mediation albumen of the present invention can also be used to screen the reagent of regulating CRC-mediation protein active.Therefore, on the one hand, characterization method of the present invention is used to identify the reagent of regulating CRC-mediation protein active, and is synthetic to CRC-mediation albumen by mediating the cell of albumen with test agent culture expression CRC-and measuring test agent, phosphorylation, function or active effect.When activating CRC-mediation albumen by phosphorylation, cultivate test agent with CRC-mediation albumen, use γ-[ ³²P]; Or [ ³³P]-ATP (or other list-nucleotide) of mark, or [ ³²P]; Or [ ³³P]-phosphate (phosphoric acid) or [ ³⁵S]-methionine, and measure the phosphorylation rate.

In another embodiment, use the cellular incubation test agent, this cell has mediated the transfection of albumen polynucleotide expression vector with CRC-, and measures the effect that test agent is transcribed CRC-mediation albumen by rna blot analysis.

In the embodiment, use the antibody of CRC-mediation albumen again, measure test agent to the synthetic effect of CRC-mediation albumen by western blot analysis.

Again in the embodiment, by using test agent, [ ³³P]-substrate cultivation CRC-mediation albumen in ATP (or other above-mentioned radio chemistry materials) and the CRC-mediation albumen approach measures the effect of reagent to CRC-mediation protein active.With all the experiment to [ ³⁵S]-cell of methionine normal labeled compares, and measures the adjusting of protein expression.Measure the speed of substrate phosphorylation by methods known in the art.The adjusting of term CRC-mediation protein active comprises excitement and antagonism.The present invention is particularly useful for screening the reagent that suppresses harmful protein active.Such reagent can be used for treatment or prevention CRC.

The invention provides the method that prevents and/or treats people CRC by the protectiveness CRC-mediation albumen of drug treatment effective dose.Preferably, mammal is the people who is in the CRC risk.

In drug screening test of the present invention, protectiveness or the harmful CRC-mediation albumen of identifying is used to identify the test compounds that can influence them and express.Therefore the test compounds of identifying is to be used for prevention, alleviates or postpone to be in the candidate therapeutic agent of the CRC outbreak of patient in the risk.

The test treatment compound that influences CRC-mediation protein expression can be, but is not limited to, and is selected from nucleic acid, compound, protein, element, lipid, antibody, sugar, isotope, carbohydrates, preparation, lipoprotein, glycoprotein, enzyme, but at least a in detector probe and antibody or its fragment, or its arbitrary composition, can the same with labelled antibody it can be detected ground mark, as described herein.Such mark includes, but are not limited to, enzyme labeling, radioactive isotope or radioactive compound or element, fluorescent chemicals or metal, chemiluminescence compound and bioluminescent compound.

In drug screening of the present invention test, identify the treatment compound, make to prevent from or postpone to be in to produce disease of patient outbreak in the CRC risk by its ability of inducing or improve protective protein to express.Can also prevent or the ability that reduces harmful protein expression is identified the candidate therapeutic compound by it, make to prevent from or postpone to be in to produce disease of patient outbreak in the CRC risk.

Can have target cell as the treatment nucleic acid of treatment compound but be not limited at least a following result of treatment: suppress transcribing of detrimental protein dna sequence dna; Suppress the translation of detrimental protein RNA sequence; Inhibition is corresponding to the RNA of harmful albumen or the reverse transcription of dna sequence dna; The posttranslational modification of Profilin matter; Induce transcribing corresponding to the dna sequence dna of protective protein; Induce translation corresponding to the RNA sequence of protective protein; Induce corresponding to the RNA of protective protein or the reverse transcription of dna sequence dna; Translated nucleic acid is protein or enzyme; And nucleic acid is introduced in the chromosome of the target cell that is used for the treatment of nucleic acid composing type or transient expression.The result of treatment of treatment nucleic acid can include, but are not limited to: close dcc gene or handle its expression, as antisense RNA or DNA; Suppress virus replication or synthetic; Gene therapy is as expressing the heterologous nucleic acid or the defect correcting albumen of coding treatment albumen; Modify RNA, as hnRNA, mRNA, the defective of tRNA or rRNA or expression are not enough; Coding medicine or prodrug, or enzyme, this enzyme produce the compound as medicine or prodrug in pathology of expressing CRC-mediation albumen or peptide or normal cell; And any other known result of treatment.What also comprise among the present invention is by providing coding protection CRC-to mediate the gene therapy of the polynucleotide of albumen.The present invention further comprises the method that the polynucleotide of the harmful CRC-mediation of the inhibition by effective dosage albumen expression in vivo prevent CRC.

In methods of treatment of the present invention, will treat compound and deliver medicine to people patient for a long time or fast.Optional, with the protective protein long term administration, in conjunction with effective dose to be different from the compound of the approach effect for the treatment of compound.

Methods of treatment of the present invention can be in conjunction with the methods of treatment of other CRC or in conjunction with handling this class method for cancer.

Described in following examples, the HEP who is derived from CRC patient and normal healthy controls personnel's colon is cultivated under the standard culture condition.

After hatching 20hr under the standard cell lines condition of culture, [ ³⁵S]-methionine exists down cell marking 4 hours.Isolated protein from cell and nutrient culture media.Come analysing protein samples by 2-D gel electrophoresis and mass spectrum.

For analyzing gel, be used to identify the protein that changes expression, in each experiment, use colon epithelial cell to carry out single experiment.This allows to make up the composograph of each condition of culture.Therefore, contrast in this way and the comparison between the cancer cell and statistical study make can identification of cancer cell in and secrete a histone matter to the nutrient culture media, these protein are compared with control case and are significantly raised or reduce.

Experiment

Propose following embodiment, how to prepare and use protein of the present invention so that provide to those skilled in the art, the complete disclosure and description of gene and test, and be not intended to the invention scope that the restriction inventor thinks.Carried out making great efforts to guarantee the accuracy (for example, content, temperature etc.) of numeral of using, but the sum of errors deviation of some experiments should be described.

Unless otherwise noted, umber is a weight portion, and molecular weight is a weight-average molecular weight, and temperature is a centigrade degree, and pressure is to be in or near atmospheric pressure.

The sign of the protein of embodiment-colorectal cancer mediation

Reagent

The chemical substance that is used for lysis buffer and level pad is: urea (ICNBiomedicals), thiocarbamide (Fluka), chaps (Sigma), DTT (Sigma), SDS (Serva).Dulbecco ' s improvement Eaglr nutrient culture media (D-MEM) (41966-029/052) and Hank ' s balanced salt solution (HBSS) (14175-053) from GIBCO ^TMBe used for the RNA enzyme A (cat.no.RAF LS005650) of DNA enzyme/RNA enzyme buffer liquid and DNA ' s I (cat.no.DPFF LS006330) from Worthington.Be used to measure the scintillating liquid of isotope introducing from Canberra Packsrd.Fixing pH-gradient band pH4-7, pharmalytes 3-10 (code No.17-0456-01), IPG damping fluid 6-11 (code No.17-6001-78) and [ ³⁵S]-methionine is from Amersham Pharmacia Biotch.The acrylamide that is used for two-dimentional gel is from Genomic Solutions.Be used for the dye reagent (test of Bio-Rad protein, catalogue No500-50006) and the N of protein determination, N '-methylene-two-acrylamide is from Bio Rad.The knot rectum is normal/and cancerous tissue obtains from Vejle hospital, as the part of normal treatment surgical procedure.

Material is collected

Operation is transferred to sample pathology department after taking out immediately.Open sample there and take out sample and be transferred to and contain transhipment medium (D-MEM from tumour/normal mucosa, its dialysis human serum, 1g glucose/L and microbiotic penicillin/streptomycin, but wherein do not have methionine remain on 37 ℃) different test tubes in.

The biopsy mark

From Vejie hospital on-carrier's colon biopsy tissue (normal/cancer) 37 ℃ of transhipment media, and use HBSS rinsing 3 times.Then with 1mm ²The section biopsy and some transfers are moved in the mark medium (dialysis human serum, 1g glucose/L, [ ³⁵S]-D-MEM of methionine).Remaining bit organization alive is put into liquid nitrogen and is stored in-80 ℃.After 24 hours, from each hole, collect the radioactivity nutrient culture media of using at 37 ℃ of marks, and will be with the media transfer of crossing to the 1.5mL Eppendorf pipe of mark.Culture medium in 37 ℃ of incubators then.With biopsy washed twice in HBSS; Collect wash solution then, be used for centrifugal loose cell and the tissue collected.After this, remove HBSS, and biopsy is transferred to dish hollow and in the dry hole, and be transferred to once more in the ice-cold homogenizer.

Protein Extraction

With 100 μ l DNA enzyme/RNA enzyme buffer liquid (25 μ g/ml RNAse A solution, 25 μ g/ml DNAse I solution, 30mM NaCl, 20mM Tris.HCl (pH7.5), 5mM CaCl ₂, 5mM MgCl ₂) add in the ice-cold homogenizer contain biopsy and 30 seconds of even matter.After 5 to 10 minutes, even once more matter.This process repeats 30 minutes up to homogenize in a organized way.Take out 150 μ l homogenate and put into clean pipe.Use 100 μ l H ₂O washs homogenizer, and inhales and move twice, and water also is taken out in the pipe.Test tube is put into liquid nitrogen and is full of nitrogen.After this test tube being put into freeze-dryer spends the night.300 μ l lysis buffers (7M urea, 2M thiocarbamide, 2%CHAPS, 0.4%DTT, 1%v/v pharmolite 3-10, IPG damping fluid 6-11) are added in the pipe and in the room temperature shaken overnight.

Protein and CPM measure

To be suitable for using the Bradford method (Bradford MM.1976) of lysis buffer to be used for the protein concentration of working sample.In triplicate, come the preparation standard curve by 10 μ l lysis buffers being added in a series of disposable plastic cuvettes that contain 0,5,10 and 40 μ g standard proteins (BSA).With H ₂O adds in each cuvette the cumulative volume until 1600 μ l.10 μ l of each sample are added 1590 μ l H in the cuvette in triplicate ₂Among the O.Then 400 μ l dye reagents are added in each cuvette and thoroughly and mix.Measure the absorption value of each sample in 30 minutes adding dye reagent at the 595nm place.Then by standard curve determination protein concentration (Fey SJ etc., 1997).

Use following method to measure the isotope of introducing in the protein.Get 5 μ l supernatants of each sample in duplicate and add among the 45 μ l 0.02%BSA.After in little test tube, mixing, add 1ml 10%w/v TCA and came precipitating proteins in 30 minutes 4 ℃ of placements.Use then the HAWP filter (HAWP 0200, Millipore, Bedford, MA, USA) by suction strainer collecting precipitation thing, filter is wetting in advance with the 1%BSA among the 10%w/v TCA.With the dry filter of hair-dryer and be transferred in the scintillation vial.Add the 4ml scintillating liquid then and place 2h at least.Use scintillation counter counting sample (Canberra Packard 2000) (Fey SJ etc., 1997) then.Gel electrophoresis

Isolated protein in first dimension on 18cm IPG4-7 band.Being used for IPG4-7 band rehydration damping fluid is lysis buffer.All samples is added to 200 μ l, this is applied to the ipg strip band and places 18 hours to reexpand.After this, add more 100 μ l lysates and washing sample therein, place 5.5 hours (Adelina Rogowska-Wrzesinka etc., 2001).With 5 * 10 ⁶CPM is loaded on each gel.

In the enterprising line focusing of Multiphor II, use total Vh 50,000 (linear increment gradient, 0V to 600V continue 2:15h, and 600V to 3500V continues 8h, remains on 3500V then, continues 9:25 hour) at 20 ℃.Focus on once finishing and band was vibrated 15 minutes in level pad (6M urea, 30% glycerine, 2%SDS, 1%DTT, 50mM Tris-HCl pH8.8) and be stored in-80 ℃.Before being loaded into second dimension, with band balance 15 minutes again in fresh level pad.

In second dimension, go up use 12.5%w/v acrylamide gel (acrylamide: bisacrylamide ratio 200: 1) carry out SDS PAGE gel electrophoresis at vertical Investigator 2-D electrophoresis system (Millipore).Being provided with down at 20 ℃ at steady current gel, race glue spends the night.To run the recycle of glue damping fluid.

The protein pattern is observed and Computer Analysis

After the bidimensional gel electrophoresis separates, with gel convection drying on filter paper, be exposed to phosphorus imager flat board (AGFA) 10 days, and AGFA ADC70 reader (Morstel, Belgium) the middle reading.Then in the corner with radioactive ink with the gel mark and be exposed to film (AGFA, Gurix RP2) 25 days, so that preparation cutting mask and identification of proteins (Nawrocki A etc., 2001).

The 2D gel pattern of using Bioimage 2-D analyser (based on UNIX's) analysis to obtain from ADC70 phosphorus imager.Use identical parameter to find the spot border to all gels.In some cases, manual synchronizing border (for example, separately too near so that the spot that is difficult to distinguish) for program.(integrated optical density IOD) is measured protein expression (Nawrocki A etc., 2001) as the summation of all pixel values in the spot border.Percent of total (%IOD) as institute's spottiness in the gel provides (Adelina Rogowska-Wrzesinka etc., 2001).With all images with reference to image (from analysis before) compare.Will above exist in 5 images and in the reference image non-existent spot be added on the correction position with reference to image.

The coupling number and the %IOD that take passages each spot also put all data and data before together.Data are divided into two groups: normal cell and cancer cell.Calculate the standard deviation of each spot in each group, and the comparative result of the standard deviation between the normal and cancer tissue of each spot square is used for ensuing student ' s t-check.

To each spot calculate student ' s t between two groups check disclose express significantly different protein (level of signifiance 99%, p=0.01).Select significantly different protein then.

Preparation property gel

For passing through the mass spectrum identification of protein subsequently, except 3 * 10 ⁶The CPM/ gel [ ³⁵S]-protein of methionine mark, load 300 μ g/ gel cryoproteins matter and run preparation property IPG4-7 gel, use identical gradient and program.After second dimension, will preparation property gel drying and be exposed to X-ray film 10 days (Adelina Rogowska-Wrzesinka etc., 2001).

Identification of proteins

The X-ray film that launches is placed on the dry gel, use the mark on the turning to come localizing objects albumen.Place and be used as the cutting mask when hyaline membrane is reorientated protein and removed the X-ray film.Directly excise protein from gel then.By at H ₂Washing is removed remaining white filter paper and by the traditional vacuum drying, is become white from the teeth outwards until it among the O from the back side of gel.Make gel at 50mM NH then ₄HCO ₃, 5mM CaCl ₂, reexpanded 45 minutes at 4 ℃ in the 12.5ng/ μ L trypsase.Supernatant does not contain tryptic same buffer by 5-10 μ L and substitutes and make it in 37 ℃ of digestion spend the night (Fey SJ etc., 1997) then.After gel digestion, analyze (Jensen ON etc., 1998) by the MALDI mass spectrum.The mass spectrum (Fey SJ etc., 1997) that uses trypsase autopepsia peptide to come internal calibration to obtain.Then they are used to search for ncbi database, by MASCOT MS/MS ion search ( Http:// www.matrixscience.com).

Following parameter is used for database search, use less improvement: mammal, trypsinization, the oxidation that methionine is possible allows a division of missing, there is not protein quality and p1 restriction, peptide tolerance ± 50-70ppm, MS/MS tolerance ± 0.5Da, peptide electric charge+1, single isotope, MALDI-TOF-TOF is used method.

" significantly " result can think in conjunction with the protein position in the evaluation of search SWISS-PROT database and the summary 2D gel pattern.From ncbi database ( Http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=Protein), the SWISS-PROT database ( Http:// www.ncbi.nlm.nih.gov/entrez/query. Fcgi? db=Protein) and the publication recited protein function of other references.

The result

In this research, mark is from colon cancer patient's normal and cancer colon biopsy tissue and analyze by 2DGE after Protein Extraction.Obtain normally/the cancer biopsy from 33 patients, and use 65 samples (a cancer sample is subjected to the pollution of bacterium) altogether.Use the protein group of each sample of IPG4-7 gel analysis.

The assistant images analysis that uses a computer detects and mates 2,128 spots.To then [ ³⁵S]-the protein 2DGE pattern of methionine mark is divided into two groups: normal and cancer.In " normally " group, there are 33 patterns, in colorectal cancer vivisection group, have 32 patterns.The example that has shown gel images among Fig. 1.

Fig. 1 shown from normal person colon (left side) and colorectal cancer biopsy [ ³⁵S]-the 2DGE pattern of the protein of methionine mark.Normal structure was from 65 years old women, and the colorectal cancer biopsy was from 59 years old male sex.

After this carry out comparison and statistical analysis between these two groups.298 protein spots demonstrate the remarkable change between two groups.They all cut from IPG4-7 preparation property gel, and accept the MALDI mass spectrum.Up to now, 66 spots have been identified, more well afoot.

In these spots, 25 spots in the cancer obtain raising, and 41 spots in the cancer obtain downward modulation.31 spots are low-density (average %IOD＜0.05 in normal and cancer group).This is 47% of whole spots of identifying.Show and to identify many weak spots.In the 2DGE pattern of the position display of identification of protein in Fig. 2, and in table 1, provided the quantitative of these protein.

Fig. 2 show from normal person's colon biopsy tissue [ ³⁵-S]-the 2DGE pattern of the protein of methionine mark.The IPG4-7 gradient is used for first Separation of Proteins of tieing up, and the SDS-PAGE of 12.5% polyacrylamide is used for second dimension.All titles are accession number of the protein identified in the Swiss-prot database.Some may be posttranslational modification albumen, some may be bigger protein and decompose fragment or posttranslational modification albumen that some are to be reported in to have the different protein of expressing or having thought the diagnostic flag that colorectal cancer is possible in people's colorectal cancer.' A ' after the protein accession number, ' B ', ' C ' ... be associated with ' A ' in the table 1, ' B ', ' C ' ..., be used for different %IOD.

Possible posttranslational modification

In more than two spots, find the protein of ten kinds of evaluations.Wherein, keratin 1 (being labeled as * among Fig. 2) is accredited as six spots very far away each other.It has been generally acknowledged that keratin 1 is the pollutant in the mass spectrum process.Therefore, these spots need to analyze once more.

In them six are accredited as two or have spots in two very approaching mutually row.Think that this spot pattern is a kind of type of posttranslational modification, changed the p1 of protein, but molecular weight does not have big change.

Fig. 3 has shown the amplification 2DGE pattern of possible PTM spot: I-plastin (9a); Intelectin (9b); Keratin 19 (9c); Ig α chain C fragment (9d); Actin, tenuigenin 2 (9e); Fibrinogen γ chain precursor (9f)

For example, I-plastin (Q14651) is accredited as two spots, and all reduces in the colorectal cancer biopsy.Neutral relatively more acid amount is bigger.Phosphorylation is the PTM of prediction, and we have confirmed this conclusion (Fig. 2 and 3a, table 1) by the speckle displacement in the 2DGE pattern.

Intelectin-1 (Q8WWA0) is accredited as two spots, and all reduces in the colorectal cancer biopsy.The N-glycosylation is the PTM (Swiss-prot) (Fig. 2 and 3b) that identifies.

Keratin, I type cytoskeleton 19 (P08727) is accredited as four spots, and all reduces in the colorectal cancer biopsy.Wherein two spots are very near (Fig. 2 and 3c).Known angle albumen is treated phosphorylation, so phosphorylation can be the PTM of prediction.

Ig α-1 chain C zone (P01876) is accredited as three spots, all reduces in the colorectal cancer biopsy.Wherein two spots are very near (Fig. 2 and 3d).Cys-352 is the protein modification site, and formation non-reducing thioether in reaction back is crosslinked between chromophore and come-at-able halfcystine.

Actin, tenuigenin 2 (P63261) is accredited as three spots.Wherein raise in the colorectal cancer biopsy near (Fig. 2 and 3e) two very much each other, another is reduced in the colorectal cancer biopsy.

Fibrinogen γ chain precursor (P02679) is accredited as two spots, and all raises in the colorectal cancer biopsy.The sulphation of C-end tyrosine has been predicted as PTM, and it can improve the compatibility to fibrin ferment, and fibrin ferment is to cause fibrinogen to be converted into fibrinous enzyme (Fig. 2 and 3f).

Five kinds of identification of proteins are two or three spots (Fig. 2 mark) that are arranged near relatively position.They may be bigger protein and its decomposition fragments.Owing to used the quick sample preparation procedure at this, we think that these decompose fragment and produce in biopsy, be important therefore.

Also will be wherein three kinds (P08727 P63261) thinks posttranslational modification for P14625, P05787, and it has changed the p1 of protein, but molecular weight does not have big change.

P08727 and P63261 below have been discussed.Endoplasm plastin precursor (P14625) is accredited as two rises and a spot downward modulation (Fig. 2) in the colorectal cancer biopsy, and of downward modulation obtains identifying in protein mixture Spot C, so protein expression may raise in the colorectal cancer biopsy.Do not report the protein modification of this albumen.

Keratin, II type cytoskeleton 8 (P05787) are accredited as three approaching spots mutually, and whole three spots are downward modulation (Fig. 2) in the colorectal cancer biopsy.The phosphorylation of Ser-73 plays an important role in the thread body weight new organization of keratin.

Protein mixture

Three spots are accredited as the potpourri of two or three protein.

(sequence covers: 32% and 22%) for Spot A:P35527 and P04264.

Spot B:P13667, (sequence covers: 23%, 33% and 24%) for P02679 and P08238;

(sequence covers: 17% and 10%) for Spot C:P14625 and P04217; (Fig. 2)

Usually, a kind of composition has better covering and higher matching value.Other compositions may be the decomposition products than larger protein, and the peptide quality covers the fragment (SpotB) of protein sequence, or may they all be decomposition products (Spot C) than larger protein, maybe may relate to some pollutants, as keratin (spot A).By not producing these fragments in the specimen preparation process at random, because having statistics between two groups, all selected spots significantly change, two groups have altogether above 60 samples.In other words, these catabolites specificity in tissue or cancer biopsy produces.

Protein function

Can the protein of identifying be divided into groups by different functions or approach.If do not mention especially, all data obtain from the Swiss-prot database.In 47 protein, 25 albumen are structural proteins, and it surpasses half (53%) of all identifying albumen.This is very easy to understand, because in the tumour production process, can destroy or change the normal configuration of cell, and for example, the cytoskeleton that can change cell influences ion channel.In addition, 23% (11) are the protein that relates in the cellular metabolism.For example, Galectins-1, secretagogue and cathepsin D work in cell differentiation or the Cycle Regulation at apoptosis.In case tumour produces, their expression must be affected.5 kinds of albumen are transport proteins, are cyto-architectural changes to this a kind of explanation, for example, film, the passage of the more inside and outside albumen transportations of cell is blocked, and this has influenced the expression of transport protein.Vice versa.4 kinds of albumen relate to immunity/system of defense.In case may be because the unusual cytoactive of immune system recognition produces as tumour, more immunocyte infiltration colons and rectum stop it.To have more multi-functional albumen such as seralbumin and put into the group that to represent its major function.For more detailed content, referring to table 2.

The relation of protein and colorectal cancer

Based on science report before, the protein of identifying can also be divided into four groups:

Reported the protein of comparing and in people's colorectal cancer, having different expression or thought the possible diagnostic flag of colorectal cancer with normal knot rectal tissue.(group A)

Reported the protein of comparing and in other cancers (that is breast cancer), having different expression or thought possible diagnostic flag with normal tissue.(group B)

Reported the protein that has with other disease association.(group C)

The protein of not reporting about the correlativity of they and any disease.(group D)

The protein title, subcellular location, in the normal and cancerous tissue of knot rectum on average %IOD list in the table 1.

The A of selection group then (having reported the protein of comparing and in people's colorectal cancer, having different expression or thought the possible diagnostic flag of colorectal cancer) with normal knot rectal tissue.Each protein will further be discussed in this group in aborning function of colorectal cancer and effect.Wherein, 3 is structural proteins, and 3 is that transport protein and 3 albumen relate to cellular metabolism.5 albumen are reduced in the colorectal cancer biopsy, and 4 albumen raise.

The albumen of downward modulation

The L-plastin

The L-plastin is a kind of isomeric form (isoform) (the T-plastin is another kind of isomeric form) of plastin, plastin is ABP's a family, it is a great expression (Lin CS etc., 1993) in the mammalian cells that guard and in all normal replication in eucaryote is evolved.It is expressed in hematopoietic cell, can obtain phosphorylation therein and have been found that in pernicious people's cell that the non-hematopoiesis of many types is originated synthetic.Because its high expressed in the human tumor cells of entity tumor, the L-plastin may work in tumour takes place or L-plastin gene be activated (Lin CS etc., 1993) in the process that tumour produces.To human prostate, breast and other steroids control bands have carried out some researchs (Lin CS etc., 1993, Zheng J etc., 1997), and the result shows that being expressed in the cancer of L-plastin raise.May be that hormone receptor has been subjected to inducing the expression of regulating the L-plastin.

Colon carcinoma cell line SW480 and the SW620 that is derived from single patient carried out another experiment.The result shows and compares in early days, and the L-plastin is (Otsuka M etc., 2001) of overexpression in the colorectal cancer late.Unfortunately, in this research, do not carry out comparison between normal and the cancer knot rectum cell.

In our research, compare with normal structure in interesting ground, being expressed in the cancerous tissue of L-plastin reduced.Because this albumen has significant change above between two groups of 60 samples altogether, can not produce arbitrarily.Average " cancer "/" normally " ratio of %IOD is 0.55, and this is very important equally.This possible explanation is that the L-plastin expression in the colon is different from the steroids control band, as breast, and prostate, ovary etc.A kind of possibility is it from normal structure to colorectal cancer biopsy is downward modulation, raises to cancer of late stage in early days from cancer.If a kind of explanation may be the brush border of small intestine be normal growth-this growth faster than infantile tumour, it will take place.But this need be by proving the cancerous tissue of different phase rather than the further research of clone.

Transgelin

Transgelin is gelation of deformation susceptibility actin and transforming protein, finds in fibroblast and smooth muscle usually (Lawson D etc., 1997).It and actin filament directly act on, and make its quick-gelatinizing in polymerization.Actin binding site or functional gelation site are relevant with the amino acid residue of cluster positive charge.The activity of Transgelin is subjected to the control of ionic strength.Lose its ability fully at high ionic strength.Its gelation activity is controlled in the formation of low ionic strength by dimer or oligomer.In the formation of actin gel, it has played boundling or crosslinked effect.Because transgelin is in the cytoskeletal organizationization of Normocellular cell and play a part importantly like this in activating, can in the carcinogenicity cell transformed, find the expression of its downward modulation.In these cells, the reorganization of cytoskeleton and unusual mobile be the key character (Shapland C etc., 1993) that shifts.Confirmed this point (Lawson D etc., 1997) by the expression of studying the fibroblast transgelin in many clones.

Some people's colon and expression of the transgelin in the mammary tumor tissue of studying investigator's colon tumor cell system and being derived from the patient have been carried out.Transgelin in showed cell system and the tissue expresses and loses or reduce (Shields JM etc., 2002) as a result.The gene expression dose that is lost in of Transgelin obtains regulation and control.Its downward modulation is expressed directly to be activated by Ras and is caused.This is important because whole human cancers about 30% in have the sudden change of Ras gene.In addition, in some tumour cells, the Ras-independent mechanism has identical functions.In other words, we we can say the downward modulation of transgelin and the transient expression relevant (ShieldsJM etc., 2002) of carcinogenic Ras.In our data, compare with normal knot rectal tissue, the transgelin in the tumor tissues is obviously reduced.Average " cancer "/" normally " ratio of %IOD is 0.71, and because 2DGE goes up the high-load that shows, it should be the good early diagnosis mark that is used for people's colorectal cancer, may be after cancer forms.In addition, because the existence of anti--transgelin, it can obtain detecting.

Liver fatty acid-in conjunction with albumen

Liver fatty acid-binding protein is the member of the fatty acid of low molecular weight protein (LMWP) in conjunction with group, and it relates to the intracellular transport (Lawire LC etc., 2004) of long-chain biologically active fat acid.It obtains specific expressed (Yamazaki T etc., 1999) in liver cell and enterocyte.Because its lipid is in conjunction with feature, it plays an important role in distributing in the dissolving of fatty acid and born of the same parents.This makes fatty acid be easier to processing (Storch J, Thumser AE.2000) in cell absorption and the born of the same parents.It also relates to fatty acid Mediated Signal Transduction approach and gene expression regulation.Therefore, it is in some important physiological function such as cell division, and growth plays effect (LawrieLC etc., 2004) in the control of differentiation etc.In the process of tumour generation and progress, these functions all obtain less regulation and control, and demonstrate optimum growth by the people embryo in the process of its normal development.

Lawrie LC expresses consistent the losing in the colorectal cancer that studies show that carry out to the liver fatty acid-binding protein in the colorectal cancer, and losing of it is early stage incident (Lawrie LC etc., 2004) in the cancer development.

In one aspect of the method, in the patient of the hepatic metastases of suffering from the colorectal cancer of being derived from, the survival year number of liver fatty acid-binding protein positive patient is higher than liver fatty acid-binding protein negative patients (Yamazaki T etc., 1999) behind the hepatectomy.This downward modulation that demonstrates liver fatty acid-binding protein is relevant with the development of tumour.

Our data accurately demonstrates normal knot rectal tissue to the identical downward modulation of colon cancer tissue and expresses trend.And average " cancer "/" normally " ratio of %IOD is 0.51.In addition, the protein content that demonstrates on the 2DGE is quite high.This makes it be easier to obtain detect.Therefore, liver fatty acid-binding protein can be the extraordinary prognostic markers of colorectal cancer.

Keratin, I type cytoskeleton 20

Keratin 20 was the cytoskeletal proteins (Schwerer MJ etc., 1996) in the gastrointestinal tract epithelial cell, and it at first obtained detecting (Moll R etc., 1992) in 1992.It is a kind of in " soft keratin " of being called who belongs to intermediate filament family.Soft keratin contains more than more than 20 members, and is divided into two main groups: I type (K9-20, acidity), II type (K1-8, neutral to alkalescence) (Zhou Q etc., 2003).

In keratin family, keratin 20 has uncommon distribution, and has to research (Zhou Q etc., 2003) seldom up to now.For example, human keratin 20 has some specific features:

Compare with other I type keratin, it only has 58% identical amino acid with keratin 14 (maximally related keratin) in conservative alpha-helix " bar " domain usually; (Moll R etc., 1993)

Its distribution almost completely is limited to stomach, enteric epithelium, urothelium, bladder, Merkel cell etc.; (Moll R etc., 1993)

It is located along crypts fine hair differentiation axle in the expression of the enterocyte that more breaks up

It lacks the immune cross-reactivity (Zhou Q etc., 2003) to most of antikeratin antibody

Its expression improves along with the differentiation in the well differentiated tissue.The correlativity between diagonal angle protein 20 and the cancer has been carried out many researchs.Verified it be important (Wildi S etc., 1999) for the sign of primary and metastatic human gland cancer.In addition, think that it is an aid mark of distinguishing cancer primary source, especially for the gland cancer (Miettinen M, 1995) of separating intestines and stomach and parenteral route source.Other studies show that out with normal colon sample and compare that CK20 mRNA has reduced (WildiS etc., 1999) in the colorectal cancer sample some that colorectal cancer clone is carried out.Think that also keratin 20 is marks of the micro-lymphatic metastasis in the colorectal cancer Central Asia and is confirmed by K-RAS that K-RAS is the gene (Yun K etc., 2000) that suddenlys change in about 30-40% colorectal cancer.

In our research, to compare with normal knot rectal tissue, the keratin 20 in the tumor tissues is reduced, and has 0.4 average " cancer "/" normally " ratio of %IOD.Yet the average content of detected protein is low relatively, and is difficult to use the protein of disappearance to be used for diagnosis, therefore can only be used to distinguish tumor type, and can be used as the auxiliary diagnosis mark of people's colorectal cancer.

Apolipoproteins A-I

Apolipoproteins A-I is the primary structure albumen of high-density lipoprotein (HDL) (HDL), and it has 234 residues.It belongs to the classification of commutative apolipoproteins, and contains spherical aminoterminal domain and lipid in conjunction with c-terminus domain (Segrest JP etc., 2000; Marcel YL etc., 2003).

Its lipid helps to have formed delicate balance (Marcel YL etc., 2003) between the lipid stability transhipment in the ability of the lipoprotein that is separated into ingredient and circulation in conjunction with feature.This has promoted cholesterol to flow out from cell, and should mechanism play an important role in supportint cell cholesterol homeostasis (Segrest JP etc., 2000).In addition, it activates lecithin in conjunction with lipid, forms the efficient that can influence reverse cholesterol transport with the ability of interactional ripe HDL of special receptor and lipid transfer protein.And and HDL together, and their effects in reverse cholesterol transport have determined the retrocorrelation (Frank PG etc., 2000) between HDL blood plasma level and the coronary heart disease.

Except coronary heart disease, reported that apolipoproteins A-I is important in other diseases, other diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and atherosclerotic (Hyka N etc., 2001).

Shown that apolipoproteins A-I expresses in human intestinal epithelial's cell.One of carrying out of Normanno studies show that and uses Apo A-I RT-PCR test healthy people or there is not can not to detect among the patient of colon cancer an apolipoproteins A-I mRNA (4 healthy donors and 11 suffer from the individualities of the tumor disease beyond the colon cancer), and find that a small amount of colon cancer patient is positive (Normanno N etc., 1998).In our analysis result, detected protein average content is very low in colorectal cancer biopsy and " normally " group, although average " cancer "/" normally " ratio of %IOD very well (0.32).What Normanno this can be interpreted as can not detect it in his normal sample.Simultaneously, the sample number of the research of Normanno N is relatively little, and can think two positive findingses in 20 accidental.To a kind of hypothesis of reducing in the colorectal cancer is in the process of cancer development, and losing of apolipoproteins A-I may destroy the homeostasis that cholesterol flows out by chance, upset cellular metabolism.The research after but such hypothesis needs confirms.

The albumen that raises

Cathepsin D

Cathepsin D is the member of histone enzyme family, and it has effect (Adenis A etc., 1995) in the extracellular matrix degraded.It is the aspartic acid endo protease that spreads all over distribution in lysosome.Cathepsin D synthesizes the 52kDa precursor protein at first on rough surfaced endoplasmic reticulum (RER), and does not have enzymatic activity after this manner.By proteolytic treatment, it splits into the active double chain form (Talieri M etc., 2004) of 46kDa.The major function of cathepsin D is the protein in acid pH degraded lysosome.In addition, in some specific cells, it can form reactive protein (Liaudet-Coopman E etc., 2005) by the precursor of removing non-activity.

Now, the cathepsin effects of discussing at most are related with cancer.Several reports have shown that the expression of this kind of enzyme is directly related with various organ cancer patients' prognosis.This is because the ability of its irritation cancer cell proliferation.The generation of cathepsin D has improved the invasion potential of cancer cell, causes the raising of metastatic potential.In addition, its can degrade extracellular matrix and activate some other think the proteinase relevant with tumour progression, as cathepsin B and L (Oh-e H etc., 2001).Yet Liaudet-Coopman E and colleague's result of study demonstrates by the unidentified cell surface receptor of direct or indirect initiation, and cathepsin D can be used as the extracellular matrix of alternative proteinase in conjunction with albumen.It not only can irritation cancer cell proliferation, and stimulate tumor-blood-vessel growth.In addition, think by with the interaction of M6P/IGFII-acceptor (it is being known by the effect in the endosome approach transhipment different ligands), cathepsin D has promoted tumor cell proliferation (Liaudet-Coopman E etc., 2005).Some other researchs have shown that cathepsin D is the crucial positive medium of induction type apoptosis.Therefore, cathepsin D may have double action (Liaudet-Coopman E etc., 2005) in apoptosis.

This data with us is consistent, compares with normal structure, and cathepsin D is overexpression in cancerous tissue.Average " cancer "/" normally " ratio very good (1.7) of %IOD is although the protein content in colorectal cancer biopsy and " normally " group is relative low.Some other reports also point out it is that cathepsin D's activity rather than content are relevant with the malignant progression of colorectal cancer.Increase continuously with content and to compare, active tumour cytocol from normal mucosa to pairing increases progressively, and when not excessive tumour becomes bigger, is (Tumminello FM etc., 1995) of successively decreasing.Therefore, the content of conjunctive tissue proteinase D albumen and active change can be given us clearer diagnostic evidence.

Galectins-1

Galectins-1 belongs to the Galectins family of the protein of being correlated with on the structure that is distributed widely in the live organism.This family is by having at least one carbohydrates-recognition structure territory and the compatibility of the oligosaccharides that contains beta galactose being limited (Danguy A etc., 2002; Rabinovich GA, 2005).

As galactose-binding protein, proved Galectins-the 1st, effective mitogen of various cell types, and many adjusting and controlling growth phenomenons have been belonged to Galectins-1.For example, by with the interaction of cell beta galactose glycosides part, it can promote mitogenesis or cell to suppress to reply.It can suppress the propagation of many cell types with the ligand interaction of the unknown, and cell comprises and combines irrelevant normal and tumour cell with the beta galactose glycosides.In addition, although this effect of some tumour cell opposings to growth and transfer, growth and survival (Scott K etc., 2004) that growth inhibited that Galectins-1 mediates in the tissue around their or apoptosis can influence tumour cell.

Reported in many human organs the overexpression of Galectins-1 in cancer cell of comparing with normal cell, organ such as bladder, thyroid gland, colon etc.In addition, reported the remarkable increase of tenuigenin Galectins-1 in the progression of disease process of human colon carcinoma.Another important observation is that the expression of Galectins-1 raising is relevant with the change of the interior position of born of the same parents of its expression.In the evolution of tumour, that sees in its expression and the normal colonic tissue in the tenuigenin appraises and decides the position increase (Hittelet A etc., 2003) gradually that becomes of comparing.Another research also points out to tie Galectins in the mucous membrane of rectum-1 overexpression relevant with tumour progression (Sanjuan X etc., 1997).Therefore, Galectins-1 is key effect (HitteletA etc., 2003) for the pernicious behavior of colon cancer cell.Our data accurately demonstrate identical expression trend, have average " cancer "/" normally " ratio of 1.61 %IOD.Therefore, Galectins-the 1st, goodish diagnosis of colorectal carcinoma mark.

Reported that another the member-Galectins-3 in the galictin family also relates to the malignant progression of colon cancer, and may be better clinical diagnosis mark (Hittelet A etc., 2003).

Proteasome 5 type α subunits

Proteasome is the high conservative that exists in all eukaryotic tenuigenin and the nuclear, a large amount of many catalysis (multicatalytic) multienzyme complex (Zevrski I etc., 2005; DaltonWS, 2004).Its major function is to eliminate cell protein, not only removes damage and albumen misfolding, and removes various albumen with important cells effect, important cells effect such as regulation of Cell Cycle, cell differentiation, apoptosis, stress response etc.Reported the degraded (Dalton WS, 2004) that is subjected to proteasome above 80% all cells albumen.Because the key effect of these substrates in cellular metabolism, so proteasome is the important component in the cell.

Effect that proteasome suppresses that the research that nuclear Factor-Kappa B (NF-κ B) is carried out is verified, it causes the stable and accumulation of its unfavorable substrate.NF-κ B is retained in the transcription factor in the tenuigenin with I-κ B (inhibition chaperone) when combining.When I-κ B does the time spent through modulated serine phosphorylation, it is degraded in proteasome.In case discharge from I-κ B, NF-κ B is transferred in the nuclear, and its control relates to cell differentiation, the open gene of growth and apoptosis then.In addition, it promotes cell proliferation and tumour to take place.The inhibition of the I-κ B degraded by proteasome inhibitor can remain on NF-κ B in the tenuigenin, therefore prevents itself and the interaction of nuclear DNA.Because the inhibitor of I-κ B phosphorylation can not prevent cell proliferation fully, this shows that the effect of proteasome inhibitor is multiple goal approach (Mitchell BS, 2003; Zavrski I etc., 2005).Bortezomib, a kind of reversible boric acid dipeptides proteasome inhibitor is first kind of proteasome inhibitor testing in the cancer clinical trial.

Therefore, although be new, proteasome is various cancers, comprises that colorectal cancer treats goodish target.(SW48, SW116) research with the colon cancer sample has proved that all proteasome inhibitor can cell growth inhibiting and apoptosis-induced (Hochwald SN etc., 2003) to colon carcinoma cell line.

According to the known cytosis of mentioning before, the rise of the proteasome in our research can be represented the development of colorectal cancer.

Seralbumin

Human serum albumins is the general transport protein in the blood.Its major function is to carry the hydrophobic fat acid molecule in blood flow.It is also in conjunction with many other natural materials and various drug molecules.

Protein is made of single polypeptied chain.It is 67% alpha-helix and does not contain β-schistose texture.It is made of three similar homeodomains, and its assembling forms heart-shaped molecule (He XM etc., 1992).

Owing to distributing widely and may reflecting the ability (Knekt P etc., 2000) that nutrition condition and protein are taken in, think seralbumin and various diseases to comprise that cancer is relevant.

Knekt studies show that with colleague carry out the higher danger coefficient of higher seralbumin level and distal colorectal intestinal cancer is relevant, but uncorrelated with the proximal colonic or the carcinoma of the rectum.In our data, it also raises in colorectal cancer, has average " cancer "/" normally " ratio of 1.82 %IOD.A kind of explanation be in the tumour and tumour around high protein take in or other dietary factors cause the seralbumin (Knekt P etc., 2000) of high concentration.

In nine kinds of albumen, wherein seven kinds have and report identical expression trend before.For remaining two kinds of albumen, L-plastin and apolipoproteins A-I, do not report between the normal and cancerous tissue of knot rectum expression ratio.In other words, for the mark of colorectal cancer, our data are in full accord with disclosing before.

Except these 9 kinds of albumen, we have identified 57 kinds of other protein that never interrelated with colorectal cancer, and propose most of or all relevant with colorectal cancer.

The conclusion that draws based on above-mentioned experimental section

In whole 298 kinds of protein of being selected by 2DGE, 125 kinds in these are raised, and are potential diagnostic flags therefore.Use 2DGE and MS, identified 66 kinds of protein that between normal knot rectal tissue and colorectal cancer tissue, have remarkable differential expression.Reported before that nine kinds in these protein served as a mark, target or to the knot rectum have influence.

This has proved that 2DGE is to find the outstanding method of colorectal cancer mark or target protein in conjunction with the MS technology.In 9 kinds of protein that discovery and discovery are relevant with colorectal cancer in these researchs, our data are consistent with all observations before and expanded all observations before.Nine kinds of albumen having reported before being not only, and all albumen of identifying also can be used for diagnostic purpose.Therefore, the result who is obtained has very high value in clinical setting.

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Claims (32)

1. diagnose the method for mammal such as people's colorectal cancer, this method comprises the existence of mensuration from least a labelled protein in the described mammiferous biological sample, does not exist or expression, and wherein labelled protein is selected from down group:

The protein that limits in the table 1; With

Be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

2. set up the expression (labelled protein of rise) that at least a labelled protein improves according to the process of claim 1 wherein that this method comprises, this labelled protein is selected from

Protein that limit and mark " rise " in the table 1; With

Be to limit the trim of protein or the protein of derivant in the table 1, make with table 1 in the protein that limits have at least 80% sequence homology.

3. according to the method for claim 1 or 2, wherein biological sample is selected from urine, ight soil, blood, saliva, lymph liquid and tissue.

4. according to the method for claim 3, wherein tissue is taken from colon and/or rectum.

5. measure mammal such as the tendentious method of people's colorectal cancer, this method comprises that mensuration exists from least a labelled protein in the described mammiferous biological sample, does not exist or expression levels, and wherein labelled protein is selected from down group:

The protein that limits in the table 1; With

Be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

6. measure the tendentious method of people's colorectal cancer, this method comprises:

A) expression that at least a labelled protein improves in the biological sample of mensuration from the people, described labelled protein is selected from down group:

I) upregulated protein that limits in the table 1 and

Ii) be i) in the trim of protein or the albumen of derivant, make and i) protein have at least 80% sequence homology;

B) expression that at least a labelled protein reduces in the biological sample of mensuration from the people, described labelled protein is selected from down group:

I) down-regulation protein that limits in the table 1 and

Ii) be i) in the trim of protein or the albumen of derivant, make and i) protein have at least 80% sequence homology; Or

C) a) and b) in the combination measured.

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

7. according to each method of claim 1,5 or 6, wherein at least a labelled protein is selected from down group:

A) with respect to impinging upon one or more protein that exist with remarkable lower or remarkable high level from the protein polyacrylamide gel of described biological sample;

B) non-existent one or more protein on the polyacrylamide gel of the protein of contrast from existing on the polyacrylamide gel of the protein of described biological sample; With

C) do not exist on the polyacrylamide from the protein of described biological sample and one or more protein of existing on the polyacrylamide of the protein that contrasts.

8. the method for treatment mammal such as people's colorectal cancer comprises the expression that changes at least a labelled protein, and this labelled protein is selected from down group:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

9. the method for treatment mammal such as people's colorectal cancer comprises being selected from following compound administration in the people with at least a:

A) protein that limits in the table 1;

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology;

C) have and a) and/or b) in the protein of protein identical function;

D) coding a), b) or c) in the nucleotide sequence of protein;

E) a), b) or c) in the antibody of protein;

F) can be in conjunction with a), b) or c) in the nucleic acid fragment of protein; With

G) can be in conjunction with a), b) or c) in the compound of protein,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

10. prevention or postpone for example method of people's colorectal cancer outbreak of mammal comprises following material is delivered medicine to the people:

A) protein that limits in the table 1;

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology;

C) a) and b) in limit the analogies of protein;

D) coding a) and/or b) in the nucleotide sequence of protein;

E) a) and/or b) in limit the antibody of protein;

F) can in conjunction with a) and/or b) nucleic acid fragment of protein; And/or

G) can in conjunction with a) and/or b) in limit the compound of protein,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

11. measure reagent has the result of treatment possibility in the colorectal cancer treatment method, comprise mensuration from one or more following albumen before test model is exposed to described reagent and relative expression's level afterwards:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

12. measure the method for compound effect in the colorectal cancer treatment, comprise the protein expression level of mensuration from one or more following albumen:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

13. measure the method for compound used therefor effect level in the colorectal cancer treatment, comprise mensuration from one or more following albumen before test model is exposed to described reagent and expression afterwards:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

14. measure mammal such as the character of people's colorectal cancer or the method for inducement suffer from or easily suffer from colorectal cancer, comprise that foundation is from the expression of following protein with respect to model:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology,

Collateral condition be when at least a labelled protein be HSP90,60S acidic ribosomal protein and/or during from the protein of table 1 group A, from this group selection another labelled protein at least, it is not HSP90,60S acidic ribosomal protein or from the protein of table 1 group A.

15. be selected from following pure basically protein:

A) protein of table 1; With

B) and a) protein has the protein of at least 80% sequence homology in,

Collateral condition is to get rid of HSP90 and 60S acidic ribosomal protein from protection.

16. comprise in the coding schedule 1 protein that limits or have the nucleic acid fragment of nucleotide sequence of the protein of at least 80% sequence homology with it.

17. with according to the nucleic acid fragment of claim 16 or its part or with the nucleic acid fragment of its complementary strand hybridization.

18. be used to detect the purposes of the existence that is selected from following peptide according to each nucleic acid fragment of claim 16-17:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology.

19. can be in conjunction with the antibody that is selected from following protein:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology.

20. according to the antibody of claim 19, it is a polyclonal antibody.

21. according to the antibody of claim 19, it is a monoclonal antibody.

22. be used to detect the purposes of the existence that is selected from following protein according to each antibody of claim 19 to 21:

A) protein that limits in the table 1; With

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology.

23. be used to diagnose mammal such as people's colorectal cancer or colorectal cancer genetic predisposition's testing cassete, comprise:

A) combination tool, its specificity is selected from following labelled protein in conjunction with at least a:

I) protein that limits in the table 1; With

Ii) be i) in the trim of protein or the protein of derivant, make to have at least 80% sequence homology with it; Or this combination tool is the antibody of at least a described labelled protein, can be in conjunction with the nucleic acid fragment of at least a described labelled protein, or can be in conjunction with the compound of at least a described labelled protein;

B) instrument that detects combination tool and at least a labelled protein or combine with at least a nucleic acid fragment, if there is combination, perhaps for detection in conjunction with the instrument of level and

C) be used for related instrument, combining of related and described combination tool, perhaps related if there is combination in conjunction with level, whether represent that individual mammal has the possibility or the trouble colorectal cancer genetic predisposition of significantly higher trouble colorectal cancer.

24. measure the method for material effect, this method comprises uses mammal, people for example, it has used to be established as according to each method of claim 1 to 7 has high likelihood of suffering from colorectal cancer or the individuality of suffering from the colorectal cancer genetic predisposition, and this method comprises the effect that material is delivered medicine to individuality and measure this material.

25. pharmaceutical composition, it comprises:

A) can regulate the material that nucleic acid fragment is expressed, this nucleic acid fragment coding is selected from least a portion of following protein:

I) protein that limits in the table 1; With

Ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology;

B) be selected from following labelled protein:

I) protein that limits in the table 1; With

Ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology;

C) be selected from the derivant of following protein, homologue or analogies: i) protein that limits in the table 1 and ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology;

D) be selected from the antibody of following protein: i) protein that limits in the table 1 and ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology;

E) can be in conjunction with the nucleic acid fragment that is selected from following labelled protein: i) protein that limits in the table 1 and ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology; And/or

F) can be in conjunction with the compound that is selected from following labelled protein: i) protein that limits in the table 1 and ii) be i) in the trim of protein or the protein of derivant, make and i) protein have at least 80% sequence homology.

26., as medicine, as be used for the treatment of or prevent colorectal cancer according to the pharmaceutical composition of claim 25.

27. be selected from the purposes of following compound:

A) protein that limits in the table 1;

B) be the trim of table 1 protein or the protein of derivant, feasible protein with table 1 has at least 80% sequence homology;

C) have and a) and/or b) in the protein of protein identical function;

D) coding a), b) or c) in the nucleotide sequence of protein;

E) a), b) or c) in the antibody of protein;

F) can be in conjunction with a), b) or c) in the nucleic acid fragment of protein; With

G) can be in conjunction with a), b) or c) in the compound of protein, be used to make the medicine of treatment or prevention colorectal cancer.

28. the cell of at least a albumen of construction expression or the method for clone, this albumen is selected from the albumen from table 1, and the trim of table 1 albumen and derivant make protein with table 1 (for example 90% or 95%) homology that has at least 80%; For example, by at least a dna sequence dna of encoding said proteins is introduced in the cell, as the cell of self.

29. according to the structure cell of claim 28 or the method for clone, wherein modifying cell, to avoid by immune system recognition be allogenic material.

30., wherein introduce the activity that at least a controlling element is regulated the dna sequence dna of introducing according to the structure cell of claim 28 or the method for clone.

31. according to the structure cell of claim 28 or the method for clone, wherein cell is a colon epithelial cell, rectum epithelial cell, stem cell or multipotential cell.

32. the cell or the clone that can obtain by the method for claim 28.

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