CN102584978A - Marker for diagnosis and prognosis of colorectal cancer, breast cancer and pancreatic cancer - Google Patents
Marker for diagnosis and prognosis of colorectal cancer, breast cancer and pancreatic cancer Download PDFInfo
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Abstract
The invention discloses a marker for diagnosis and prognosis of colorectal cancer, breast cancer and pancreatic cancer. A human voltage gated proton channel Hv1 is taken as a marker; the colorectal cancer, the breast cancer and the pancreatic cancer are diagnosed by using methods such as immunohistochemistry and the like; and the Hv1 is taken as an index for judging prognosis. A gene sequence and an amino acid sequence of the Hv1 are as shown in a sequence table SEQ ID No.1 and SEQ ID No.2. The Hv1 is highly expressed in tissues of the colorectal cancer, the breast cancer and the pancreatic cancer, and is not expressed in tissues of normal and innocent tumors. The expression level of the Hv1 in the tissues of the colorectal cancer, the breast cancer and the pancreatic cancer is related with the tumor malignization degree, and the expression level of the Hv1 in the tumor with high malignization degree is high. Meanwhile, the expression level of the Hv1 in the tissues of the colorectal cancer, the breast cancer and the pancreatic cancer is related with is related with the survival rate of patients, and the patients with high expression level of the Hv1 are bad in prognosis and high in death rate. Therefore, the Hv1 has broad application prospect and potential social benefit as the marker for diagnosis and prognosis of the colorectal cancer, the breast cancer and the pancreatic cancer.
Description
Affiliated field
The invention belongs to the biologic medical technical field, relate to a kind of new colorectal cancer, mammary cancer and carcinoma of the pancreas mark, its essence is the valtage-gated proton channel Hv1 in people source.This invention can be widely used in the diagnosis of colorectal cancer, mammary cancer and carcinoma of the pancreas and prognosis.
Background technology
Cancer is the general designation of one big type of malignant tumour, and the disease that cause not normal by control growth and proliferation of cell mechanism.Cancer cells except grow out of control, healthy tissues even transfer to other parts of health arround also can local invading via body-internal-circulation system or lymphsystem.Because the happening part of various cancers is different, pathomorphism is different, and developmental stage is different, therefore can produce various clinical manifestations.But cancer in early days often symptom seldom just show a series of sings and symptomses gradually after waiting to develop into certain phase.Generally performance and two aspects of general symptom are treated by the clinical manifestation office of being divided into of cancer.The characteristics of cancer cells be unrestrictedly, hyperplasia without end, make the intravital nutritive substance of patient by mass consumption; Cancer cells discharges multiple toxin, makes human body produce a series of symptoms; Cancer cells also can be transferred to whole body growth and breeding everywhere, causes that human body is become thin, unable, anaemia, poor appetite, heating and serious organ function be impaired or the like.
China has become one of multiple state of malignant tumour at present.Malignant tumour comprises cancer and sarcoma, and the malignant tumour of being originated by the epithelium cell is called cancer; Malignant tumour by the mesenchymal tissue source is called sarcoma.Malignant tumour can occur in all age group.Along with the increase at age, the sickness rate of cancer can increase gradually, and especially its sickness rate of the elderly more than 40 years old significantly increases; Sarcoma is higher at children and young adult's sickness rate.Malignant tumour is not only the life that threatens the patient to the mankind's harm, returns misery, stress and economical load that the patient brings body.
Cancer diagnosis mode commonly used at present has following several kinds: the image check at certain position can help the doctor to judge whether that tumour exists in the image check, health.The X ray examination is doctor's a most common form, like rabat, X ray examination bone etc.Also have some special X ray examinations, like CT scan: be connected with the X-X-ray machine X with a computingmachine, can draw the intravital detailed image of a series of bodies.Magnetic Resonance Imaging (MRI) is connected a strong electro-magnet with computingmachine, can draw with CT scan similar but the effect pictures different.Radionuclide scanning, the patient is oral or inject a kind of slight radioactive material that has, and by the radioactive rays level of some organ of scanning device measurement, being analyzed by the doctor then has the dense abnormal area that gathers of no radiation in certain organ.UW is the other a kind of method of internal structure of having a physical examination.This high frequency sound wave that can not be heard by the people gets into health, and bounce-back is returned then, and this just forms a ultrasonoscopy.These pictures can be presented at one on the screen of televisor, also can on paper, print.Splanchnoscopy, during splanchnoscopy, the doctor directly observes a very thin pipe through throat, tracheae, esophagus, enteric cavity, vagina or uterus, and the while can gather unusual tissue or cell carries out pathological analysis.Laboratory examination, the laboratory examination of blood and urine examination etc. can be the doctor provides important information.If cancer is arranged in the health, the level of some material also can become unusual in the human body.Detect blood, cerebrospinal fluid, stool and urine and phlegm etc., the existence that can point out some tumour through the laboratory.Whether check pathological section, health check-up, image check, endoscope and laboratory examination can show has abnormal conditions to exist in the health, be cancer but have only check pathological section to make a definite diagnosis at last.In cut sections for microscopic examination, the doctor gets a fritter tissue sample or excises whole tumour from unusual place.Pathologist is observed at microscopically.Exist if confirm cancer, it is the cancer of what kind that pathologist can be told usually, and can judge this growth of tumour cell speed.Therefore, seek the biomarker of malignant tumour, for diagnosing cancer, especially early diagnosis cancer and judging prognosis have very big help.This mark must be in healthy tissues be seldom expressed, and in cancerous tissue a large amount of specifically expressings, not only can be used to diagnosing cancer or judging prognosis, assistance wait by stages, can also be used as the target site of immune guiding treatment simultaneously.We can say that the biomarker of cancer all plays an important role at aspects such as the diagnosis of malignant tumour, prevention, treatment, judging prognosis.
Colorectal cancer is the general name of the colorectal carcinoma and the rectum cancer, and colorectal cancer is meant the malignant change prognosis mala that big epithelium of intestinal mucosa takes place under multiple carcinogenic factor effects such as environment or heredity, and mortality ratio is higher.Colorectal cancer is the malignant tumour of big epithelium of intestinal mucosa origin, is one of modal malignant tumor of digestive tract, and incidence is only second to cancer of the stomach and esophagus cancer.
Colorectal cancer is in different areas, and its sickness rate has obvious difference.According to world's epidemiology investigations of tumor statistics, economically developed national sickness rate is higher, and the sickness rate of country variant differs 60 times.Colorectal cancer in the North America, the sickness rate on ground such as West Europe, Australia, nz is the highest, ground such as Japan, Chile, Africa are then low.At western developed country, colorectal cancer is the second malignant neoplasm that is only second to lung cancer.Show that according to pertinent data as if the sickness rate of African colorectal cancer very low.In general, worldwide China belongs to the low district of sending out.Colorectal cancer is equally on the rise with lung cancer in recent years, and China is no exception.This disease sickness rate at home also has areal variation, is the district occurred frequently with Shanghai, Zhejiang, Fujian.
In China's common cancer death, colorectal cancer patients accounts for the 5th the male sex, and the women accounts for the 6th.The sickness rate of male sex's colorectal cancer is about 1.6: 1 apparently higher than the women.Recent two decades comes the sickness rate of colorectal cancer increasing gradually, simultaneously, and its age of onset trend aging.Predilection site is rectum and rectum and sigmoid colon intersection, accounts for 60%.Sickness rate age aspect data according to domestic statistics, was many with 40～50 years old, and the age group median is about 45, about 1/3 of the whole cases of person below 40 years old, and person below 30 years old accounts for about 10%.The national large bowel cancer age occurred frequently occurred frequently is 60～70 years old, and person below 30 years old accounts for about 6%.China's colorectal cancer was sent out age age ratio well 10～15 years old ahead of time abroad, and person below 30 years old accounts for 11%～13%, and this is a principal feature of China's colorectal cancer.
Seek the biomarker of colorectal cancer, for the diagnosis colorectal cancer, especially early diagnosis colorectal cancer and judging prognosis have very big help.This mark must be in healthy tissues be seldom expressed, and in colorectal cancer tissue a large amount of specifically expressings, not only can be used to diagnose colorectal cancer or judging prognosis, assistance to wait by stages, can also be used as the target site that immune guiding is treated simultaneously.We can say that the biomarker of colorectal cancer all plays an important role at aspects such as the diagnosis of colorectal cancer, prevention, treatment, judging prognosis.
The biomarker that is usually used in detecting colorectal cancer at present is mainly by following several kinds: (1) S-CEA (CEA) is measured: in colorectal cancer patients serum; Can detect CEACAMS (CEA); This is a kind of gp; Often coming across in the malignant tumor patient serum, is not the special related antigen of colorectal cancer, so change of serum C EA measures this sick diagnosis is not had specificity.But detect CEA with radioimmunology, quantitatively dynamic observe, surgical effect and the monitoring postoperative recurrence of judging colorectal cancer had the certain significance.After like the colorectal cancer underwent operative tumour being excised fully, change of serum C EA then descends gradually; If recurrence can raise again once again.(2) CA19-9 is a kind of tumor markers of glycoprotein analog, and content obviously raises in the serum of Infusion in Patients with Digestive.Change of serum C A19-9 concentration and Duke are proportionate by stages, and along with upgrading concentration rising by stages, so the size of concentration has clinical value for the prognosis of judging colorectal cancer.But it is lower that serum detects the positive rate of CA19-9, only has 22.39%, divide at each Duke interim, serum with organize in positive rate difference condition and the CEA of CA19-9 identical.Duke A～Duke C is interim, and the positive rate that detects CA19-9 through serum all is starkly lower than the postoperative tissue, and especially Duke A phase patient, the result who detects change of serum C A19-9 is negative.CA19-9 is not high to the early diagnosis value of colorectal cancer, but the observation of curative effect of colorectal cancer is had important clinical significance with the recurrence monitoring.(3) CA50 is present in cytolemma with fat or lipoprotein bonded form, belongs to sphingoglycolipid class affinity tag, is the mark of pancreas and colorectal cancer.
Follow a series of molecular biological changes in the generation of colorectal cancer, the development, the ideal tumor marker should have higher susceptibility and specificity, and can react the progress and the result of treatment of tumour.Yet still do not have a kind of tumor markers can satisfy above-mentioned condition so far, therefore seek all more satisfactory mark of a kind of susceptibility, specificity, especially, have broad application prospects having vital role aspect the colorectal cancer early diagnosis.
Mammary cancer is one of modal malignant tumour of women, and its sickness rate accounts for the 6.7%-14.9% of women's malignant tumour, is only second to uterus carcinoma.About 50% case in underwent operative treatment back can be cured, and all the other cases of 50% possibly recur or shift.
Mammary cancer be the breast glandular epithelium under multiple carcinogen effect, transgenation has taken place, cause hyperplasia out of control.Because change has taken place in the biological behavior of cancer cells, and causes cell to demonstrate unordered, unconfined neoplasm.Cancer cells infinite multiplication and the unordered shape ground that its histology takes the form of a large amount of primitivizations crowds agglomerating, the NBT's structure around extruding and the erosion damage.
Because it is free that breast cancer cell is easy to come off, and sends out whole body with blood or lymph liquid etc., form early stage far-end and shift, cause its clinical cure difficulty.The transfer of whole body important organ such as lung transfer, brain transfer, bone transfer etc. are all with direct threats people's life, so mammary cancer is seriously to jeopardize the malignant disease of human life.
Some specific antigenic materials that breast cancer cell exists have very big help for the detection of mammary cancer.Whether through the detection for these marks, just can obtain biopsy or specimens from pri is mammary cancer.
The markers for breast cancer that has been found that at present has tens kinds, they or directly related with people source mammary cancer endocrine, maybe can assist breast cancer diagnosis, or relevant with Prognosis in Breast Cancer.These antigens comprise: ERs (estrogen receptor ER), progesterone receptor (progesterone receptor PR); Androgen receptor (androgen receptor AR); The PS2 gene is claimed mammary cancer estrogen-induced gene (breast cancer estrogen induced again; BCEI), CA
15～3Antigen, and CEACAMS (carcinoembryonic antigen, CEA), lactotropin (prolactin, PRL), tumor susceptibility gene (BreastPovarian cancer susceptibility gene, BRCA
1), the p53 cancer suppressor gene, and PCNA (proliferating cell nuclear antigen, PCNA); C-erbB-2 (HER-2/neu) gene, and cathepsin D (Cathepsin-D, Cath-D); The p27 cancer suppressor gene, the p21 cancer suppressor gene shifts cancer suppressor gene nm23 etc.
Routine clinical is undesirable to judging in long term of mammary cancer by stages, need set up newly for the clinical method that directive significance is arranged, and research shows that some marks of reflection knubble biological behavior can be judged the prognosis of mammary cancer more accurately.
So we we can say, the mark of mammary cancer plays an important role at aspects such as the diagnosis of mammary cancer and judging prognosis.Certainly, these marks are special more the expression of breast cancer tissue or cell surface, and its is just more accurate and obvious with the effect in the prognosis in diagnosis.
In sum, seek a specific specificity high, in mammary cancer The positive expression rate high, be positioned at the breast cancer cell surface, the biomarker that the people had antigenic mammary cancer has crucial meaning for the diagnosis and the prognosis of human breast carcinoma.
Carcinoma of the pancreas is one of digestive tube common malignancy, is modal in the malignant tumour, and pilosity is born in head of pancreas portion.Stomachache and painless jaundice are the common sympton of carcinoma of head of pancreas.The long-term a large amount of smokings of diabetic subject, higher fatty acid high animal protein drink trencherman, sickness rate increases relatively, and this disease is mainly in the elderly, and the male patient is many far beyond premenopausal women, and the postmenopausal women's sickness rate and the male sex are similar.Pathogenic factor it be unclear that, and finds that a little environmental factorss are relevant with the generation of carcinoma of the pancreas.Fixed primary Hazard Factor are smoking, mellitus chololithiasis drink higher fatty acid high protein diet of feed such as (comprising beer) and chronic pancreatitis and purified flour foods, and gastrectomy also is the generation risk factor for pancreatic cancer, its mortality ratio is high.
Nineteen forty-three, Rockeg has at first carried out total pancreatectomy.Domestic surplus civilian light was at first reported the case of head of pancreas duodenum excision in 1954.In recent years, the sickness rate of carcinoma of the pancreas rises year by year, is 9.0/10 ten thousand at the U.S.'s 1988 annual morbidities, the man: the woman is 1.3: 1.Be more common in person more than 45 years old.Sweden's sickness rate is higher, is 1,25/,100,000, and remains unchanged in 20 years in the past.Britain and Norway have respectively increased by 1 times.Compare with the sixties seventies, and the mark sickness rate of Canada, Denmark and Poland has increased more than 50%.In China, carcinoma of the pancreas has become one of ten big malignant tumours of human mortality.The carcinoma of the pancreas patient that BJ Union Hospital admits to hospital has in recent years increased by 5～6 times than the fifties.And according to the 354 routine analysiss of cases of Beijing area 7 tame hospitals, 41～70 years old person accounts for 80% among the patient, and in recent years, young carcinoma of the pancreas patient is also than the trend that obvious increase was arranged before 10 years, and grade of malignancy is higher, and prognosis is poorer.With regard to the happening part of carcinoma of the pancreas, see at most with the head of pancreas position that still account for about 70%, body of pancreas takes second place, tail of pancreas portion more takes second place, and a body afterbody that has all has, and belongs to diffuse lesion or multicentricity pathology.
Find that at present the tumor markers relevant with carcinoma of the pancreas has kind more than 10, clinical commonly used except that CA19-9, CA50, Span-1, Dupan-2, POA, CEA etc. in addition.Mostly domestic used reagent is import at present, and expensive, most hospitals still fail to popularize.But the detection of tumor marker is to the monitoring of screening, diagnosis postoperative recurrence and the transfer of carcinoma of the pancreas, and the discriminating of pancreas innocent and malignant tumour all has vital role.If the tumor marker inspection with two or more can improve its positive rate.1, CA19-9 is the related antigen of carcinoma of the pancreas, and its expression depends on the expression of lewis blood group antigen, the lewis negative patient, and the inspection of CA19-9 is also negative.Susceptibility, specificity and the accuracy of CA19-9 diagnosis of pancreatic cancer are respectively 83.1%, 73%, 75%; And the level of little carcinoma of the pancreas patient CA19-9 is not high; Less than the carcinoma of the pancreas positive rate of 2cm only 60.7%, and the level of the bigger patient CA19-9 of tumour is higher, and positive rate reaches more than 80%; Cut off value is decided to be 120kU/L, is higher than this value person and highly suspects carcinoma of the pancreas.Simultaneously can also judging prognosis, CA19-9 reduces to normal value behind the tumor resection, and the serum threshold value is that 30kU/L person's prognosis is better, if obviously the raising once again of tumor recurrence, transfer or sb.'s illness took a turn for the worse visible CA19-9.So CA19-9 is the significant notation thing of carcinoma of the pancreas.2, CA50 also is an antigen of lewis system, with CA19-9 good dependency is arranged, and needn't simultaneously these two antigens be measured together.But if CA50 and CA242 joint-detection can be improved the susceptibility to diagnosis of pancreatic cancer.3, Span-1 carcinoma of the pancreas patient can be up to 1446KU/L.Its susceptibility is 81%, and specificity is 68%, but undesirable for the diagnosis of little carcinoma of the pancreas, is merely 56% less than the tumer positive rate of 2cm.4, its threshold value of Dupan-2 is 400KU/L, is 50%～70% to the susceptibility of carcinoma of the pancreas, can make positive rate bring up to 95% with the CA19-9 joint-detection, and courage receives the influence of liver function, has closely related with GOT.5, CEA (CEACAMS) positive rate 78.9%, obviously improves for low differentiation carcinoma of the pancreas＞3cm tumour and the carcinoma of the pancreas positive rate of clinical IV phase, and CEA negative patient mean survival time (MST) is 38.6 months, obviously prolongs than positive person, can be used for judging prognosis.
The biomarker of above-mentioned these carcinoma of the pancreas still can not extensively be carried out, and specificity is on the low side, and is lower to early cancer and little carcinoma of the pancreas (tumour of＜2cm) positive rate, remains the cross reaction in certain false positive He other tumours.Therefore, seek a kind of high specificity, the high biomarker of positive rate has great importance to the early diagnosis and the prognosis of carcinoma of the pancreas.
Summary of the invention
The present invention seeks to provides a kind of with the mark of valtage-gated proton channel Hv1 as colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis in order to solve the problem that colorectal cancer, mammary cancer and carcinoma of the pancreas lack susceptibility, specificity marker thing.
The present invention relates to a kind of valtage-gated proton channel Hv1 of mark of new people source colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis.Hv1 is high expression level in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue, in healthy tissues and innocent tumor, does not express; The clinical stages of expression level and the cancer of Hv1 in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue and patient's survival rate are relevant, so Hv1 can be used as the mark of colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis.
The mark that is used for colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis provided by the invention is the valtage-gated proton channel Hv1 in people source, and its gene is the sequence shown in the sequence table SEQ ID No.1; The valtage-gated proton channel Hv1 in mark people source of its genes encoding is the amino acid residue sequence shown in the sequence table SEQ ID No.2.
Said mark people source valtage-gated proton channel Hv1 albumen total length is 273 amino-acid residues; According to the hydrophobic property of this each amino-acid residue of passage, can Hv1 albumen total length be divided into 3 structural domain: N-end born of the same parents intracellular domain, membrane spaning domain and C-end born of the same parents intracellular domain; Hv1 has special highly selective to proton, efficiently the transmembrane transport proton.
The invention provides colorectal cancer, mammary cancer and the corresponding polyclone of carcinoma of the pancreas antigen Hv1 and monoclonal antibody, antibody fragment and verivate.The C end primer of the people source colorectal cancer that is designed, mammary cancer and carcinoma of the pancreas antigen Hv1 is shown in sequence table SEQ ID No.3 and SEQ ID No.4.
Our research shows the valtage-gated proton channel Hv1 in people source high expression level in people's colorectal cancer, mammary cancer and carcinoma of the pancreas tissue, and in normal and innocent tumor tissue, does not express basically.The malignization degree of expression level and the tumour of Hv1 in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue is relevant, and expression amount is high in the high tumour of malignization degree, expression amount is low in the low tumour of malignization degree for Hv1.Simultaneously, Hv1 survival rate of expression level and patient in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue is relevant, and the patients'prognosis that the Hv1 expression amount is high is bad, and mortality ratio is high.Therefore utilize the mark of valtage-gated proton channel Hv1, can be used for the diagnosis and the prognosis of colorectal cancer, mammary cancer and carcinoma of the pancreas as colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis.Because colorectal cancer, mammary cancer and carcinoma of the pancreas lack of specific mark are used for diagnosis and prognosis, so this mark has very wide application prospect and potential huge social benefit.
Our research shows that Hv1 plays key effect to growth, invasion and attack and the migration of colorectal cancer, mammary cancer and carcinoma of the pancreas.Suppress the expression of Hv1 in colorectal cancer, mammary cancer and carcinoma of the pancreas and obviously suppress growth of tumor, invasion and attack and transfer ability.Its mechanism is because Hv1 participates in the pH value in the regulate tumor cell, keeps stable p H value in the tumour cell.
We provide the valtage-gated proton channel Hv1 in people source mark as colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis in the present invention.Polyclone and the monoclonal antibody of utilization Hv1 detect the expression level of Hv1 in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue with methods such as immunohistochemistries, thereby diagnose and prognosis.
The invention provides with Hv1 and diagnose the method with prognosis colorectal cancer, mammary cancer and carcinoma of the pancreas; Colorectal cancer, mammary cancer and the corresponding polyclone of carcinoma of the pancreas antigen Hv1 and monoclonal antibody, antibody fragment and verivate.The present invention can be widely used in the diagnosis of colorectal cancer, mammary cancer and carcinoma of the pancreas and prognosis, has potential huge social benefit.
Advantage of the present invention and positively effect:
Mark as colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis provided by the invention, promptly valtage-gated proton channel Hv1 is a high expression level in a kind of colorectal cancer the people, mammary cancer and the carcinoma of the pancreas tissue, and in normal and innocent tumor tissue, does not express.The malignization degree of expression level and the tumour of Hv1 in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue is relevant, and Hv1 expression amount in the high tumour of malignization degree is high, in the low tumour of malignization degree, do not express.Simultaneously, Hv1 survival rate of expression level and patient in colorectal cancer, mammary cancer and carcinoma of the pancreas tissue is relevant, and the patients'prognosis that the Hv1 expression amount is high is bad, and mortality ratio is high.Therefore utilize the mark of valtage-gated proton channel Hv1, can be used for the diagnosis and the prognosis of colorectal cancer, mammary cancer and carcinoma of the pancreas as colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis.Because colorectal cancer, mammary cancer and carcinoma of the pancreas lack of specific mark are used for diagnosis and prognosis, so this mark has very wide application prospect and potential huge social benefit.
Description of drawings
Fig. 1 is the expression A (100 *) of Hv1 in colorectal cancer, B (500 *), and C (100 *) and D (500 *) are normal knot rectal tissue; E (100 *) and F (500 *) adenoma tissue; G (100 *), H (500 *), I (100 *), J (500 *), K (100 *) and L (500 *) are the colorectal cancer tissue.Healthy tissues comprises hyperplasia and adenoma, and the negative and weak positive dyes.In cancerous tissue, Hv1 shows strong positive.Hv1 dyes brown, and nucleus is blue.
Fig. 2 is the expression SW620 of Hv1 in colorectal cancer clone, HT29, and LS174T, Colo205 and SW480 cell use immunocytochemistry (A) and real-time quantitative PCR (B) to detect respectively.A.HT29 (a, 200 *), LS174T (b, 200 *), Colo205 (c, 200 *), SW480 (d, 200 *), SW620 (e, 200 *) and SW620 (f, 500 *) detect with immunocytochemistry.B. detect Hv1 at SW620 with real-time quantitative PCR, HT29, LS174T, mRNA expression level in Colo205 and the SW480 cell.All cells and SW620 are relatively.
Fig. 3 is total survival curve of colorectal cancer patient and the anosis recurrence survival curve that the relation of Hv1 expression amount and survival rate is just expressed Hv1.Low expression patient is longer lifetime than total lifetime of high expression level patient and anosis recurrence.A. total survival curve, B. is anosis recurrence survival curve
Fig. 4 is that breast cancer tissue's sample of the expression patient of Hv1 in mammary cancer is used the anti-Hv1 antibody staining.Be coloured to the feminine gender or the weak positive in normal galactophore tissue (A) and cyclomastopathy tissue (B and C) Hv1, but dyeing and strong dyeing (D-I) in breast cancer tissue, becoming.Brown, nucleus au bleu are distinguished in Hv1 dyeing.A, B, D, F and H are 200 times; C, E, G and I are 500 times.Negative control replaces immune serum with the mouse NIS.
Fig. 5 is the expression MCF-7 of Hv1 in breast cancer cell line, MDA-MB-231, and T-47D, SK-BR-3, MDA-MB-468 and MDA-MB-453 cell use immunocytochemistry (A) and real-time quantitative PCR (B) to detect respectively.A.MCF-7 (a, 200 *), MDA-MB-231 (b, 200 *), T-47D (c, 200 *), SK-BR-3 (d, 200 *), MDA-MB-468 (e, 200 *) and MDA-MB-453 (f, 200 *) detect with immunocytochemistry.B. detect Hv1 at MCF-7 with real-time quantitative PCR, MDA-MB-231, T-47D, SK-BR-3, mRNA expression level in MDA-MB-468 and the MDA-MB-453 cell.All cells and MDA-MB-231 are relatively.
Fig. 6 is total survival curve of mammary cancer carninomatosis people and the anosis recurrence survival curve that the relation that concerns Hv1 expression amount and survival rate of Hv1 expression amount and survival rate is just expressed Hv1.Low expression patient is longer lifetime than total lifetime of high expression level patient and anosis recurrence.A. total survival curve, B. is anosis recurrence survival curve
Fig. 7 is the expression of Hv1 in carcinoma of the pancreas.A (100 *) and B (500 *), carcinoma of the pancreas is organized negative control (replacing Hv1 antibody with Balb/c mouse NIS); C (100 *) and D (500 *) are #379689 carcinoma of the pancreas tissue; E (100 *) and F (500 *) are #380190 carcinoma of the pancreas tissue; G (100 *), H (500 *) is #380333 carcinoma of the pancreas tissue.It is strong and weak different that Hv1 dyes in the carcinoma of the pancreas tissue of different grade malignancies, and Hv1 shows strong positive in the high carcinoma of the pancreas tissue of grade malignancy, and in the low carcinoma of the pancreas tissue of grade malignancy, dyeing is the weak positive.Hv1 dyes brown, and nucleus is blue.
Embodiment
Patient and tissue samples
Central hospital's surgical operation obtains the colorectal cancer tissue samples from Tonghua, is the tissue samples of calendar year 2001 to 2007 year.These patients are not through the chemicotherapy treatment.139 routine colorectal cancer tissues and corresponding cancer beside organism use 10% formalin fixed, paraffin embedding.These patients' medical history information provides in table one.In addition, for the specificity that confirms that Hv1 expresses, we have also selected the not tissue of canceration, the normal knot of 10 examples rectal tissue, 18 routine hyperplastic tissues, and 20 routine adenoma tissues.Each patient's pathological data is all according to neoplasm staging, and tumour is big or small, lymphatic node state classification.The tumour differentiation degree is according to the Edmondson staging.
Central hospital's surgical operation obtains breast cancer tissue's sample from Tonghua, is the tissue samples of calendar year 2001 to 2007 year.These patients are not through the chemicotherapy treatment.105 routine breast cancer tissues and corresponding cancer beside organism use 10% formalin fixed, paraffin embedding.These patients' medical history information provides in table two.In addition, for the specificity that confirms that Hv1 expresses, we have also selected the not tissue of canceration, 8 routine normal galactophore tissues, 20 routine cyclomastopathy tissues.Each patient's pathological data is all according to neoplasm staging, and tumour is big or small, lymphatic node state classification.The tumour differentiation degree is according to the Edmondson staging.
The carcinoma of the pancreas tissue samples obtains from Tianjin Tumour Hospital's surgical operation, is the tissue samples in 2004 to 2008.These patients are not through the chemicotherapy treatment.152 routine carcinoma of the pancreas tissues and corresponding cancer beside organism use 10% formalin fixed, paraffin embedding.In addition, for the specificity that confirms that Hv1 expresses, we have also selected 10 routine normal pancreatic tissues.Each patient's pathological data is all according to neoplasm staging, and tumour is big or small, lymphatic node state classification.The tumour differentiation degree is according to the Edmondson staging.
Embodiment 1
One of preparation of valtage-gated proton channel Hv1 (people source colorectal cancer, mammary cancer and carcinoma of the pancreas recombinant protein antigen) at first designs the C end primer of the people source colorectal cancer, mammary cancer and the carcinoma of the pancreas antigen Hv1 that make new advances, and sequence is following:
C-Hv1 BamH I-F:5 ' CGCGGATCCAAGACACGTTCAGAACGGC (sequence 3)
EcoR I-R:5 ' CGGAATTCCTAGTTCACTTCACCAAGAAG (sequence 4)
Through pcr amplification, double digestion PCR product and carrier pGEX6p-1 also reclaim, and connect, and obtain recombinant plasmid.This clone is exactly the clone who contains the recombinant expression plasmid of valtage-gated proton channel Hv1 (people source colorectal cancer, mammary cancer and the carcinoma of the pancreas antigen) dna sequence dna that the present invention mentions, its called after pGEX6p-1-c-Hv1.PGEX6p-1-c-Hv1 is transformed into competence E.coli BL21 (DE3) intestinal bacteria, uses 0.5mM IPTG inductor, carry out protein expression.Obtain valtage-gated proton channel Hv1 albumen (people source colorectal cancer, mammary cancer and carcinoma of the pancreas recombinant protein antigen) through the IX column purification, gene order shown in sequence table sequence 1, aminoacid sequence is shown in sequence 2.
Two of the preparation of valtage-gated proton channel Hv1 (people source colorectal cancer, mammary cancer and carcinoma of the pancreas antigen)
Artificial synthetic polypeptide; Sequence is CSEKEQEIERLNKL; (or bovine serum albumin is connected by quality 1 to 1, obtains people source colorectal cancer, mammary cancer and carcinoma of the pancreas antigen (keyhole limpet haemocyanin (KLH)-conjugated Hv1 peptide CSEKEQEIERLNKL) (sequence 5) this polypeptide and hemocyanin then.
Embodiment 2:
(1) preparation of anti-people source colorectal cancer antigen Hv1 rabbit polyclonal antibody
Adopt the recombinant protein antigen of people source colorectal cancer, mammary cancer and the carcinoma of the pancreas antigen Hv1 of every embodiment of 4mg/ 1 acquisition to mix the subcutaneous immune nz of complete freund adjuvant multiple spot large ear rabbit, adopt the recombinant protein antigen of 4mg/ every people source colorectal cancer antigen Hv1 to mix the subcutaneous immunity second time of incomplete freund adjuvant multiple spot nz large ear rabbit after 10 days.After the immunity for the second time, carry out immunity for the third time at interval a week.Blood sampling in immune for the third time back 10 days is surveyed and to be tired, when tire＞1: 10
6, put to death the animal blood sampling, adopt coagulation to collect serum, low-speed centrifugal goes deposition.With saline water dilution in 1: 2, last ProteinA affinity chromatography column purification, the 0.1M glycine buffer wash-out of pH2.8 is dialysed 3 times to PBS with the back among the 1M Tris, detects OD280 and decides anti-body contg, obtains the rabbit polyclonal antibody of anti-people source colorectal cancer antigen Hv1.If tire＜1: 10
6, then continue immunity to tire＞1: 10
6
(2) anti-people source colorectal cancer antigen Hv1 mouse monoclonal antibody
New people source colorectal cancer, mammary cancer and the carcinoma of the pancreas recombinant protein antigen that adopt every embodiment of 100ug/ 1 to obtain mix the subcutaneous immune Balb/c mouse of complete freund adjuvant; Every recombinant protein antigen of 4 week back employing 50ug/ mixes complete freund adjuvant abdominal cavity booster immunization; Adopt every recombinant protein antigen of 50ug/ to mix complete freund adjuvant abdominal cavity booster immunization after 4 weeks once more; 3 booster immunizations altogether, blood sampling is surveyed and is tired after 10 days, when tire＞1: 10
6, put to death animal, collect the spleen tissue, make it be separated into unicellular through 100 purpose screen clothes.Adopt 50% PEG to urge fusion method, merge the back and adopt the HAT substratum to carry out screening and culturing 5,000,000 splenocytes and 1,000,000 SP2/0 cytogamy.Cultivate the ELISA that adopts people source mammary cancer recombinant protein antigen to encapsulate after 1 week and screen, positive colony carries out subclone continuously, screens the clone of stably excreting specific antibody, obtains the mouse monoclonal antibody of anti-people source breast cancer antigen Hv1.
Embodiment 3:
Antibody test colorectal cancer, mammary cancer and carcinoma of the pancreas with valtage-gated proton channel Hv1
Adopt routine immunization group method, paraffin section, with the lowlenthal serum sealing, an anti-mouse anti human colorectal cancer antigen Hv1 polyclonal antibody that adopts, the two anti-goat anti-mouse IgG-HRP that adopt, DAB colour developing, phenodin transfect cell nuclear.
Result such as Fig. 1, Fig. 4 and shown in Figure 7, painted intensive on cancerous tissue, organize normally simultaneously not to be colored.Prove valtage-gated proton channel Hv1 high expression level in colorectal cancer, mammary cancer and pancreatic cancer cell.
The colorectal cancer coloration result is according to the IRS method, and by two different pathologist marking, 0-3 is divided into the low expression of Hv1 respectively, and 4-12 is divided into the Hv1 high expression level.Use the SPSS17.0 statistic data, shown in table three, the expression of Hv1 and tumour size, tumor grade, the lymphoglandula state is relevant with clinical stages, and is irrelevant with other factors.
The mammary cancer coloration result is according to the IRS method, and by two different pathologist marking, 0-3 is divided into the low expression of Hv1 respectively, and 4-12 is divided into the Hv1 high expression level.Use the SPSS17.0 statistic data, shown in table four, the expression of Hv1 and tumour size, tumor grade, the lymphoglandula state is relevant with clinical stages, and is irrelevant with other factors.
Embodiment 4:
The relation of valtage-gated proton channel Hv1 and colorectal cancer, mammary cancer and carcinoma of the pancreas patient existence and recurrence
Mode through phone is paid a return visit obtains the situation that each patient is survived at present and had or not recurrence, uses the SPSS17.0 statistic data, and like table five, shown in table six and the table seven, table eight, patient's survival rate and recurrence situation and Hv1 expression level have direct relation.
Embodiment 5:
P53 in the colorectal cancer, Ki-67, TopoII, the relation between GST-π and P-gp expression level and the pathological parameter
Use known p53, Ki-67, TopoII, GST-π and P-gp antibody carry out immunostaining to the colorectal cancer tissue respectively, and the result adds up shown in the table nine.P53 relevant with tumor grade (P=0.011), Ki-67 and differentiation relevant (P=0.010), TopoII and differentiation relevant (P=0.010), GST-π and tumour size relevant (P=0.007) are with differentiation relevant (P=0.000).
ER in the mammary cancer, PR, Her-2, Ki-67, TopoII, the relation between GST-π and P-gp expression level and the pathological parameter
Use known ER, PR, Her-2, Ki-67, TopoII, GST-π and P-gp antibody carry out immunostaining to breast cancer tissue respectively, and the result adds up shown in the table ten.Her-2 expresses relevant with tumor grade (P=0.015), relevant with the lymphoglandula state (P=0.058), relevant with clinical stages (P=0.029).Relevant with tumor grade (P=0.012).TopoII and tumour size relevant (P=0.005).
Table one colorectal cancer clinical and pathological data
Table two mammary cancer clinical and pathological data
Relation in table three colorectal cancer between Hv1 expression level and other pathological data
Relation in table four mammary cancer between Hv1 expression level and other pathological data
Single factor logistic regression analysis of Hv1 expression level in table five colorectal cancer
aCI?indicates?confidence?interval.
Hv1 expresses and the multivariate Cox ratio venture analysis of anosis recurrence survival rate with total survival rate in table six colorectal cancer
aRR?and?CI?indicate?relative?risk?and?confidence?interval，respectively.
bP?values?were?obtained?by?Cox?proportioral?hazards?analysis?modeled?for?high?and?low/negative?levels?of?Hvl?expression.
Single factor logistic regression analysis of Hv1 expression level in table seven mammary cancer
aCI?indicates?confidence?interval.
Hv1 expresses and the multivariate Cox ratio venture analysis of anosis recurrence survival rate with total survival rate in table eight mammary cancer
aRR?indicates?relative?risk；
bCI，confidence?interval.
cP?values?were?obtained?by?Cox?proportional?hazards?anal?ysis?modeled?for?high?and?low/negative?levels?of?Hvl?expression.
P53 in table nine colorectal cancer, Ki-67, TopoII, the relation between GST-π and P-gp expression level and the pathological parameter
Table nine is continuous
ER in table ten mammary cancer, PR, Her-2, Ki-67, TopoII, the relation between GST-π and P-gp expression level and the pathological parameter
Table ten is continuous
Claims (4)
1. a mark that is used for colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis is characterized in that the valtage-gated proton channel Hv1 in described mark behaviour source, and its gene is the sequence shown in the sequence table SEQ ID No.1.
2. the mark that is used for colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis according to claim 1 is characterized in that the valtage-gated proton channel Hv1 in described mark people source is the amino acid residue sequence shown in the sequence table SEQ ID No.2.
3. the mark that is used for colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis according to claim 2; It is characterized in that said mark people source valtage-gated proton channel Hv1 albumen total length is 273 amino-acid residues; According to the hydrophobic property of this each amino-acid residue of passage, can Hv1 albumen total length be divided into 3 structural domain: N-end born of the same parents intracellular domain, membrane spaning domain and C-end born of the same parents intracellular domain; Hv1 has special selectivity to proton, efficiently the transmembrane transport proton.
4. according to claim 1, the 2 or 3 described marks that are used for colorectal cancer, mammary cancer and diagnosis of pancreatic cancer and prognosis, the C end primer of people source colorectal cancer, mammary cancer and the carcinoma of the pancreas antigen Hv1 that it is characterized in that being designed is shown in sequence table SEQ ID No.3 and SEQ ID No.4.
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| CN106047989A (en) * | 2015-04-08 | 2016-10-26 | 中国科学院北京基因组研究所 | Application of circular RNA to colorectal cancer inspection marker |
| CN106148494A (en) * | 2015-04-08 | 2016-11-23 | 中国科学院北京基因组研究所 | The application in colorectal cancer biomarker of a kind of circular rna |
| CN106148495A (en) * | 2015-04-08 | 2016-11-23 | 中国科学院北京基因组研究所 | The application in colorectal cancer biomarker of a kind of circular rna |
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| CN113122627A (en) * | 2019-12-30 | 2021-07-16 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of IGFL4 gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer |
| CN113122627B (en) * | 2019-12-30 | 2023-08-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of IGFL4 gene as marker in diagnosis and treatment of ovarian cancer |
| CN113088554A (en) * | 2020-01-08 | 2021-07-09 | 复旦大学附属华山医院 | Application of phosphatase PHLPP2 in preparation of marker preparation for treating pancreatic cancer prognosis through vitamin C |
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