CN102010903A - Application of IL-23 (Interleukin 23) in early pathological changes diagnosis of liver cancer - Google Patents

Application of IL-23 (Interleukin 23) in early pathological changes diagnosis of liver cancer Download PDF

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CN102010903A
CN102010903A CN 201010268466 CN201010268466A CN102010903A CN 102010903 A CN102010903 A CN 102010903A CN 201010268466 CN201010268466 CN 201010268466 CN 201010268466 A CN201010268466 A CN 201010268466A CN 102010903 A CN102010903 A CN 102010903A
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antibody
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曲春枫
杜君
陈陶阳
王金兵
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Cancer Hospital and Institute of CAMS and PUMC
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Abstract

The invention relates to a method for identifying a chronic hepatitis B virus infection object to have a liver cancer generation trend, comprising the following steps of checking an IL-23 level in a biological sample of the object, such as blood samples of blood serum or derivatives thereof and comparing the IL-23 level and an IL-23 level in a control sample, wherein in a checking sample, the raising of the IL-23 level indicates that the object has the liver cancer generation trend. The invention also relates to the application of a preparation for checking the IL-23 to the preparation of a composition which is used for identifying the object to have the liver cancer generation trend and a kit for identification.

Description

The application of IL-23 in the early hepatocarcinoma pathological changes diagnosis
Invention field
The present invention relates to the novel marker and the method for early hepatocarcinoma pathological changes diagnosis, also relate to the test kit that is used for the early hepatocarcinoma pathological changes diagnosis.
Background of invention
Primary hepatocarcinoma is one of the highest malignant tumour of China's incidence, annual number because of the death of trouble liver cancer accounts for 50% of whole world PLC mortality number, the people's life and health in serious threat, has caused heavy economical load for patient family and country.In China, the main diseases that liver cancer takes place shows as serum hepatitis B virus surface antigen lasting masculin because of being the infection of chronic persistence hepatitis B virus, and this part crowd is the population at risk that liver cancer takes place.Show according to national epidemiology survey result in 2006, among China nature crowd after the hepatitis B virus surface antigen adjustment positive rate estimate at hepatitis B virus surface antigen carrier 9,400 ten thousand people up to 7.18%.At present, single limited for the treatment means of the liver cancer of clinical diagnosis, the first place of in the majority kind of malignant tumour of mortality in said patients.Therefore, prevent chronic lasting hepatitis B virus infection person progress, find the early lesion of liver cancer early, and then take positive diagnosis and treatment measure, can greatly improve the survival rate of liver cancer patient for liver cancer.
" clinical diagnosis of primary hepatocarcinoma with standard " by stages of formulating in Guangzhou September calendar year 2001 according to " liver cancer Professional Committee of Chinese Anti-Cancer Association ", (Chinese hepatopathy magazine, the 324th page of the 9th the 6th phase of volume of December calendar year 2001), China at present the most frequently used primary hepatocarcinoma method of early diagnosis is regularly to carry out the detection of alpha-fetoprotein in the serum and the examination of iconography (high risk population: 40 years old and the above male sex, 45 years old and above women's chronic hbv-infection person) in the high risk population.Case definition is for having one of following feature at least: (a) AFP 〉=400 μ g/L, gestation, reproductive tract embryo source property tumour, reactivity hepatopathy and secondary liver cancer can be got rid of, and enlargement, hard and the liver of major tubercle shape lump or the occupying lesion person that imaging examination has liver cancer characteristic arranged can be touched; (b) AFP＜400 μ g/L, can get rid of gestation, reproductive tract embryo source property tumour, reactivity hepatopathy and secondary liver cancer, and have two kinds of imaging examinations that the occupying lesion of liver cancer characteristic is arranged or have two kinds of liver cancer markers (DCP, GGT II, AFU and CA19-9 etc.) positive and a kind of imaging examination that the occupying lesion person of liver cancer characteristic is arranged.(c) clinical manifestation of liver cancer and the outer metastatic lesion (comprise macroscopic bloody ascites or find cancer cells therein) of sure liver is arranged and can get rid of the secondary liver cancer person is arranged.And often the making after liver cancer takes place of this diagnosis, patient has lost best treatment opportunity.In addition, epidemiology survey shows, in chronic viral hepatitis B virus surface antigen carrier, has every year 1% crowd make progress naturally and is liver cancer, and the incidence of liver cancer totally has 40% approximately all the life.Therefore in the high risk population, find early stage characteristics of lesion early, thereby take positive means and measure to intervene and treat raising patient's quality of life and survival rate.Need to set up the method for efficient convenient easy row.
By the chronic viral hepatitis B progress is that liver cancer is a very long process, is 40-50, is body and the long-term results of interaction of virus.Therefore, the clinician is usually at 40 years old and the above male sex, the level of alpha-fetoprotein in its serum and the examination that additional ultrasonic image is learned regularly detect in this high-risk colony of chronic hbv-infection person of 45 years old and above women, find the early hepatocarcinoma patient in the hope of adopting existing conventional means.Recent study is found, in the patient of chronic hbv-infection, the liver cell of virus infection secretes multiple different cytokine with the immunocyte of identification infected liver cell, is bringing into play important effect in the course of disease progress that by the chronic hbv-infection status progression is liver cancer.
Interleukin 23 (IL-23) is a heterodimer hematopoietic cytokine, is made up of p40 and two subunits of p19, is mainly produced by body activatory scavenger cell and dendritic cell, can promote the propagation of T cell and the generation of IFN-γ.IL-23 preferentially stimulates body memory t cell rather than primary tape T cell mass.Its acceptor wide expression is in activating T cell, memory t cell, NK cell, scavenger cell and dendritic cell, IL-23 acts on these cells, induce releasing and activity of inflammatory cytokines before the IFN-γ etc., play a significant role in the morbidity of diseases associated with inflammation, autoimmune disorder and aspect such as antitumor and anti-infective.
Summary of the invention
In one aspect, the invention provides a kind of identify the chronic HBV infection object for example the people have the method that the liver cancer tendency takes place, comprising:
Detection derives from the level of IL-23 in the biological sample of described object, wherein in the test sample IL-23 level to raise be that described object has the indication that the liver cancer tendency takes place.
In one embodiment, described biological sample is blood sample such as serum or derivatives thereof.
In one embodiment, the level of IL-23 is determined by the level of the mRNA of detection IL-23 albumen or IL-23 in the described sample.
In one embodiment, use IL-23 specific antibody or its Fab to detect the proteic level of IL-23 in the described biological sample.
In one embodiment, in the described test sample IL-23 level to raise be that the IL-23 level is compared with IL-23 level in the control sample and raise in the test sample.
In one embodiment, the rising of IL-23 level is that the IL-23 protein level is higher than cutoff value in the test sample in the described test sample.In one embodiment, to have the cutoff value that the liver cancer tendency takes place be 855pg/ml to object.
In one embodiment, the proteic level of IL-23 is determined by detecting p19 subunit or its homologue in the described test sample.
In yet another aspect, the invention provides a kind of be used to identify the chronic HBV infection object for example the people have the composition that the liver cancer tendency takes place, wherein said composition comprises the preparation of level that the biological sample IL-23 level that is used for detecting described object for example detects the mRNA of IL-23 albumen or IL-23.
In one embodiment, described biological sample is a for example serum or derivatives thereof of blood sample.
In one embodiment, use IL-23 specific antibody or its Fab to detect the proteic level of IL-23 in the described biological sample.
In one embodiment, described preparation is at the proteic specific antibody of IL-23, for example mono-clonal or polyclonal antibody.
In yet another aspect, the invention provides IL-23 specific antibody or its Fab preparation be used for identifying the chronic HBV infection object for example the people have the application of the composition that the liver cancer tendency takes place.
In yet another aspect, the invention provides be used to identify the chronic HBV infection object for example the people have the test kit that the liver cancer tendency takes place, described test kit comprises that the biological sample IL-23 level that is used for detecting described object for example detects the preparation of level of the mRNA of IL-23 albumen or IL-23.
In one embodiment, described biological sample is a for example serum or derivatives thereof of blood sample.
In one embodiment, described preparation is at the proteic specific antibody of IL-23.In one embodiment, described antibody is mono-clonal or polyclonal antibody.
The accompanying drawing summary
Fig. 1: by the effect of experimenter's operating characteristic curve analysis and evaluation IL-23 to prediction hepatocellular carcinoma (HCC).Area under the ROC is 0.699 (95%CI 0.593/0.791) (two represented curves of dotted line is 95% fiducial interval among the figure).When cutoff value was 855pg/ml, susceptibility and specificity were respectively 60.0% and 91.1%.
Fig. 2: Kaplan-Meier analyzes the dependency that is used to assess between IL-23 level and the HCC CIR.The result shows that the CIR of HCC among the patient with high IL-23 level is higher.
Fig. 3: the sample serum of the timed interval when following up a case by regular visits to end according to distance with different time points is divided into 5 groups.The result shows in developing into the patient of HCC, and is higher in the early stage IL-23 concentration of progression of disease, reduces then, and IL-23 concentration keeps constant between follow-up period in control patients.
Detailed description of the Invention
Unless otherwise indicated, Science and Technology term used herein should have usually known implication of those skilled in the art. Unless need especially in addition, then singular references should comprise plural number, plural term should comprise odd number. Aforementioned techniques and method are carried out according to the described conventional method of list of references of quoting at this specification that reaches well known in the art usually. See and for example incorporate Current Protocols inImmunology.Wiley﹠Sons for referencial use into, Hoboken, NJ is described. All lists of references that this paper quotes comprise that patent, patent application, article, textbook etc. reach the list of references of wherein quoting and all quote adding this paper with it at this.
The inventor has set up a for a long time HBV chronic carrier formation of follow-up study in the area, Qidong of the middle and later periods nineties in last century one of in the district occurred frequently of China's hepatitis liver cancer, regularly carry out the detection of serum alpha-fetoprotein level and the ultrasonic image of liver and gall spleen and learn inspection, found some early liver cancer patients. Utilize this formation studied simultaneously in Chronic Patients with HBV Infection body for the immune response of virus by the chronic hepatitis B progress being effect in the liver cancer process, in the hope of finding to affect the serum cytokines feature of liver cancer early lesion. Result of study shows, as far back as being to adopt before existing routine clinical method is diagnosed as the 5-8 of liver cancer by chronic hepatitis progress, compare with continuation chronic hepatitis B the infected, obvious variation has just taken place in the Cytokine expression profile in the liver cancer patient blood serum, and wherein the most obvious cell factor of difference is IL-23. Adopt the testing result of the enzyme linked immunological cloth tactical deployment of troops (ELISA Array) to show that IL-23 has preferably indicative function for chronic hepatitis patient course advancement final result, can effectively carry out the early warning diagnosis to continuation chronic hepatitis B progress for the patient of liver cancer. It is the early lesion blood serum designated object of primary carcinoma of liver that IL-23 can be used as by continuation chronic hepatitis B progress.
In one aspect, the invention provides identify the chronic HBV infection object for example the people have the method that the liver cancer tendency takes place, be included in the level that detects IL-23 in the biological sample of described object, wherein to raise be that described object has the indication that the liver cancer tendency takes place to the level of IL-23 described in the test sample.
" liver cancer " described herein refers to primary hepatoma disease. Term used herein " cancer " is often referred to the malignant growth of any type, namely compares any morphology and/or the physiology change that show or have the target cell that cancer feature tendency takes place with unaffected (health) wild type control cells. The example of this change can relate to cell size and shape (become big or diminish), cell proliferation (cell number increase), Cell Differentiation (physiological status variation), apoptosis (apoptosis) or cell survival.
Term used herein " have take place cancer tendency " refers to that any to be in precancerous condition be the cell phenotype that normal cell is converted into the indication of the intermediateness in the tumour cell process. In other words, this term refers to take place the state of the risk of cancer.
The liver cancer of common type is hepatocellular carcinoma (HCC). Term used herein " hepatocellular carcinoma " refers to former liver malignancy. Most of HCC are secondary to chronic hepatitis virus infection (hepatitis B or hepatitis C) or cirrhosis (alcoholism is the common factors that causes cirrhosis). Hepatocellular carcinoma is the same with any other cancer, when existence causes the cell high speed duplicating and/or causes cell to avoid molecular mechanism when sudden change of apoptosis and take place. Especially, the chronic infection of hepatitis B and/or hepatitis C can help the generation of hepatocellular carcinoma by causing body autoimmunity system repeatedly to attack self liver cell (some of them are by virus infections, other only be the onlooker).
In one embodiment, described biological sample is blood sample such as serum or derivatives thereof. Biological sample among the present invention can be people or non-human source. Yet the present invention typically carries out with the human biology sample. Biological sample described herein is any biological sample from measurand, especially comprises the Arbitrary Samples of nucleic acid or polypeptide. Advantageously, can comprise following sample: blood, serum, blood plasma, blood platelet, saliva, phlegm, urine etc. more generally comprises any tissue, organ of nucleic acid or polypeptide or biological fluid advantageously. In addition, biological sample can contain derived from cell hepatocellular cell extract or comprise its cell mass for example. In in-vitro method of the present invention for detection of sample usually should collect in clinical acceptable mode, for example collect in the mode of protection nucleic acid or protein. In an example, described sample is the sample that is derived from blood, such as the sample of blood, serum or blood plasma. Can by any known technologies useful as by the blood sampling, obtain sample by the Noninvasive technology from sample collection point or storehouse etc. Can also the described sample of preliminary treatment increasing the accessibility of target molecule, as by cracking (machinery, chemistry, enzymatic lysis etc.), purifying, centrifugal, separation etc. Can also the described sample of mark so that the detection that target molecule exists (fluorescence, radioactivity, luminous, chemical, enzyme labeling etc.). In an example, described biological sample is whole blood sample, does not namely also pass through the blood of separating step, its optional can being diluted.
In one embodiment, the level of IL-23 is determined by the level of the mRNA of detection IL-23 albumen or IL-23 in the described sample, for example uses IL-23 specific antibody or its Fab to detect the level of IL-23 albumen in the described biological sample.
IL-23 albumen is made up of p19 and two subunits of p40; Wherein p19 (SEQ ID NO:1:MGSRAVMWTAGRAVGGS SAWTCSKCTAWSAHVGHMDRGDTTNDVHCGDGCDGRDNSCRHGYKGSDTGSDSVGH ASGSGHHWTSSSWRRKRSAVAVAARVAHGAATS) is peculiar subunit, and p40 (SEQ ID NO:2:MCHVSWSVASVAWKKDVYVVDWYDAGMVVTCDTDGTWTDSSVGSGKTTVKG DAGYTCHKGGVSHSHKKDGWSTDKDKKNKTRCAKNYSGRTCWWTTSTDTSVKSSRG SSDGVTCGAATSARVRGDNKYYSVCDSACAASVMVDAVHKKYNYTSSRDKDKNKKN SRVVSWYDTWSTHSYSTCVVGKSKRKKDRVTDKTSATVCRKNASSVRADRYYSSSW SWASVCS) is the subunit of sharing with homologue. Therefore, in one embodiment, the level of described detection IL-23 albumen can be undertaken by the level that detects p19 subunit or its homologue, and wherein the rising of the level of IL-23 described in the test sample is that described object has the indication that the liver cancer tendency takes place.
The homologue of p19 subunit refers to comprise with amino acid sequence shown in the SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the polypeptide of the amino acid sequence of higher homology.
The homology of two protein sequences is corresponding to the percentage that is positioned at the same amino acid of identical or similar position in two protein chains. Utilize BLOSUM 62 array computation percent homology with the BLAST algorithm, this algorithm can be in the NCBI website (http://www.ncbi.nlm.nih.gov/)Obtain. In this article, the homologue of described p19 subunit is functional homologue preferably, i.e. its polypeptide that has significant similitude (for example at the about 50% sequence homogeny of amino acid levels) and form with the p40 subunit with SEQ ID NO:1. At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or the functional homologue of the p19 subunit of at least about 99% sequence homogeny be preferred functional homologue.
Can be that polypeptide, nucleic acid, carbohydrate, lipid, small molecular weight compounds, oligonucleotides, oligopeptides, RNA disturb (RNAi), antisense RNA, recombinant protein, antibody or its conjugate or fusion for detection of the preparation of the mRNA of IL-23 albumen, p19 subunit or its homologue, IL-23. About RNAi, visible Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev.2003Dec; 55 (4): 629-48.Review.), about antisense RNA, visible Opalinska JB, GewirtzAM. (Sci STKE.2003Oct 28; 2003 (206): pe47).
For example, in order to determine the level of IL-23 albumen, p19 subunit or its homologue in the object, method of the present invention can be used in conjunction with the part of IL-23 albumen or p19 subunit and measure level (concentration or absolute magnitude) from IL-23 albumen in the biological sample of described object or p19 subunit or its homologue, and there is the ill risk of liver cancer in IL-23 albumen or p19 subunit or its homologue level described object of indication that raises in the described biological sample of object.
Part described herein can be receptor target material, cell factor, hormone, growth factor, receptor specific antibody and pattern recognition receptors (PRR) part. In an example, described part is antibody (or its Fab). Can utilize IL-23 albumen, p19 subunit or its homologue in any technology announcement well known by persons skilled in the art or the analytic sample, especially for example utilize ligands specific, such as antibody or fragment or antibody derivatives. Preferred described part is that the derivative of the fragment (such as Fab, Fab ', CDR etc.) of specific antibody or this antibody-like of described albumen or this antibody-like is (such as single-chain antibody, ScFv). Compound by detecting target and part as the part that utilizes mark, detector ligand of utilizing the second mark etc. can test sample in existence or the amount of target protein. Can use known immunological technique to comprise ELISA, RIA etc.
Can especially utilize the immunogen immune non-human animal who comprises described albumen (perhaps its immunogenic fragments) and reclaim antibody (polyclone) or the specific antibody of cellulation (to produce monoclonal antibody) generation target protein by routine techniques. For example in following document, describe for the preparation of polyclone or monoclonal antibody, ScFv fragment and the people's or humanized antibody technology: Harlow et al., Antibodies:A Laboratory Manual, CSH Press, 1988; Ward et al., Nature 341 (1989) 544; Bird et al., Science 242 (1988) 423; WO94/02602; US5,223,409; US5,877,293; WO93/01288.
" antibody " described herein, " Fab " or " immunogenicity part " all have usually known implication of those skilled in the art. For example " Fab " of antibody refers to produce by recombinant DNA technology or by enzyme or the complete antibody of chemical cleavage, comprises Fab, Fab ', F (ab ')2, Fv and single-chain antibody (svFc). Antibody can be polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, people's antibody and can be the antibody of mark and fragment, variant or the derivative of described antibody. Antibody labeling can be radioactive label, fluorescence labeling, enzyme labeling, chemiluminescent labeling or biotin group mark. Antibody or its preparation of its fragment and use be know and be disclosed in for example Harlow, E.and Lane, D.,Antibodies：A LaboratoryManual，Cold Spring HarborLaboratory Press，Cold Spring Harbor，New York，1999。
Many specific antibodies known in the art at IL-23 albumen, p19 subunit or its homologue, Anti-Human IL-23 (p 19) Monoclonal antibody for example, Clone hlt2736 (Biolegend), Anti-Human IL-23p 19, Clone eBio473P 19 (eBioscience) and Anti-Human IL-23p 19Functional Grade Purified, Clone HNU2319 (eBioscience).
Can also utilize the variation of known technology for detection protein expression of mass spectroscopy those skilled in the relevant art and/or structure, in general this class technology is assigned to Proteomic analysis under one's name, so that detect the specific characteristics sequence in patient's blood.
Perhaps, method of the present invention can use any detection method of the variation of the mRNA that detects IL-23 to measure the level of the mRNA of IL-23 in the described biological sample, and the level of the mRNA of IL-23 raises and indicates described patient to have the ill risk of liver cancer in the wherein said biological sample.Above-mentioned detection method comprise can the test sample amplifying nucleic acid various technology, as the Northern trace, selective cross uses bag by the base material of oligonucleotide probe for example arrayed nucleic acid molecule, DNA chip etc., by for example RT-PCR, quantitative PCR or connect nucleic acid amplification of PCR or the like.These methods can comprise that use can selectivity or the nucleic acid probe or the primer of specific detection sample amplifying nucleic acid target.For example, those skilled in the art are known and can reference standard condition (Sambrook, Fritsch, Maniatis (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) hybridize, for example can high, in or hybridize under the low stringency.Perhaps, those skilled in the art can utilize known the whole bag of tricks to increase, as the use of the amplification (TMA) of PCR, LCR, transcriptive intermediate, strand displacement amplification (SDA), NASBA, allele specific oligonucleotide (ASO), allele specific amplification, Southern trace, strand conformation analysis (SSCA), in situ hybridization (for example FISH), gel shift, heteroduple analysis or the like.
" level rising " described herein be meant by detect value that above-mentioned biological sample obtains and reference point for example in chronic viral hepatitis B the infected who does not suffer from liver cancer or normal people observed intermediate value or mean value or with based on experimental evidence and/or prior art data pin to the chronic HBV infection object for example the people have the cutoff value that the liver cancer tendency takes place and compare and raise.
In one embodiment, in the described test sample IL-23 level to raise be that the IL-23 level is compared with IL-23 level in the control sample and raise in the test sample.
Term used herein " control sample " typically is meant from having chronic HBV infection but does not have the biological sample of the object of liver cancer characteristics of lesion.The definite of the level of target typically follows the standard program of having set up well known in the art (referring to for example Current Protocols in Immunology.Wiley﹠amp; Sons, Hoboken, NJ; Current Protocols in Molecular Biology.Wiley﹠amp; Sons, Hoboken, NJ).Determine and to carry out at protein level or rna level, for example using enzyme immunoassay or Western trace method to carry out protein level detects, perhaps the RNA specific probe carries out the Northern engram analysis, perhaps carries out at dna level by quantitative PCR or real time pcr behind reverse transcription (and clone) RNA.
Difference between one or more testing sample that is obtained and the level of one or more control sample can be normalized to for example level of house-keeping gene of further contrast nucleic acid, and the level of house-keeping gene is known not according to the morbid state of cell and difference.House-keeping gene for example comprises beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1 etc.
But, replace in any experiment, determining the level of one or more control sample, also can be based on experimental evidence and/or prior art data definition one or more cutoff value at specified disease phenotype (being morbid state).
In this article, cutoff value is meant the point of positive likelihood ratio in the ROC curve each point (for the ratio of True Positive Rate with false positive rate) and negative likelihood (for the ratio of false negative rate with the true negative rate) ratio maximum.
Therefore, in one embodiment, the rising of IL-23 level is that the IL-23 protein level is higher than cutoff value in the test sample in the described test sample.In embodiment preferred of the present invention, described cutoff value is 855pg/ml.
In the present invention, term " evaluation has the tendency that the liver cell cancer takes place " also comprises prediction and probability analysis (on " diagnosis " meaning).Composition disclosed herein and method are intended to clinical application, with the decision form of therapy, comprise therapeutic or preventative intervention, Case definition such as disease stage and diseases monitoring and surveillance of disease.According to the present invention, can be provided for checking the intermediate result of Obj State.This intermediate result can be diagnosed out this object to suffer from this disease with help doctor, nurse or other practitioner with the extraneous information combination or have the risk of suffering from this disease.
Term used herein " polypeptide ", " peptide " and " protein " is used interchangeably, and refers to the polymkeric substance of amino-acid residue, and it comprises through 9 of peptide bond bonded or more a plurality of amino acid.Polymkeric substance can be linear, and branched or cyclic can comprise naturally occurring and/or amino acid analogue, and it can be interrupted by non-amino acid.Usually, if aminoacid polymers is long (for example surpassing 50 amino-acid residues), it preferably is called polypeptide or protein, but if it is 50 amino acid longs or shorter, preferably is called " peptide ".In this article, term " nucleic acid ", " nucleic acid molecule ", " polynucleotide " and " nucleotide sequence " are used interchangeably, and define the polymer of any length, as poly-deoxyribonucleotide (DNA) (cDNA for example, genomic dna, plasmid, carrier, viral genome, separated DNA, probe, primer and its any mixture) or poly-ribonucleotide (RNA) molecule (for example mRNA, sense-rna) or the poly-ribose of blended-poly-deoxyribonucleotide.They comprise strand or two strands, and are linear or circular, natural or synthetic polynucleotide.In addition, polynucleotide can comprise non--naturally occurring Nucleotide, for example methylated Nucleotide and nucleotide analog and can be interrupted by the non-nucleotide component.If exist, before or after polymerization, can carry out nucleotide modification.
In yet another aspect, the present invention also provide specific detection IL-23 albumen or IL-23 mRNA preparation preparation be used for identifying the chronic HBV infection object for example the people have the application of the composition that the liver cancer tendency takes place.In an example, described preparation is the proteic part of IL-23.Part described herein can be receptor target material, cytokine, hormone, somatomedin, receptor specific antibody and pattern recognition acceptor (PRR) part.In one embodiment, described part is antibody (or its Fab).In one embodiment, described to liking the people.At part is in the example of antibody, described antibody can be polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, people's antibody and can be the antibody of mark and fragment, variant or the derivative of described antibody that perhaps described Fab is Fab, Fab ', F (ab ') 2, Fv and single-chain antibody (svFc).
In yet another aspect, the present invention also provide be used to identify the chronic HBV infection object for example the people have the test kit that the liver cancer tendency takes place, described test kit comprises the preparation of level of the mRNA of the biological sample, for example blood sample or derivatives thereof IL-23 albumen or the IL-23 that are used for detecting from described object.Described test kit comprises the arbitrary substance that can be used to implement the method for the invention.In an example, test kit of the present invention comprises the antibody of specificity at IL-23 albumen or immunogenic fragments.
Embodiment
The present invention further illustrates by following embodiment, but any embodiment or its combination not should be understood to the restriction to scope of the present invention or embodiment.Scope of the present invention is limited by appended claims, and in conjunction with the general general knowledge of this specification sheets and this area, those of ordinary skills can clearly understand claims institute restricted portion.Under prerequisite without departing from the spirit and scope of the present invention, those skilled in the art can carry out any modification or change to technical scheme of the present invention, and this modification and change are also contained in the scope of the present invention.
Embodiment 1:
Be the patient of HBsAg male chronic viral hepatitis B virus (HBV) persistent infection from 1996 to 1999 in Qidong, Jiangsu Province recruitment at least 2 tests in 2 years.The crowd who gets rid of hepatitis C virus or HIV coinfection and other property followed chronic hepatopathys.Carry out B ultrasonic at baseline and detect to get rid of existing of liver cirrhosis and HCC, in the process of following up a case by regular visits to, nobody accepted antiviral therapy.Research approach obtains the Chinese Academy of Medical Sciences and Qidong liver cancer study on prevention institute ICR/Ethics Committee of hospital (EthicsCommittee of Cancer Institute/Hospital, Chinese Academy of Medical Sciencesand Qidong Liver Cancer Institute) approval (approval number: #09-59/354).Progress is diagnosed for " clinical diagnosis of primary hepatocarcinoma with standard " by stages that all patients of HCC were formulated according to calendar year 2001 " liver cancer Professional Committee of Chinese Anti-Cancer Association ", has at least one following standard: (a) AFP 〉=400 μ g/L, gestation, reproductive tract embryo source property tumour, reactivity hepatopathy and secondary liver cancer can be got rid of, and enlargement, hard and the liver of major tubercle shape lump or the occupying lesion person that imaging examination has liver cancer characteristic arranged can be touched; (b) AFP＜400 μ g/L can get rid of gestation, reproductive tract embryo source property tumour, reactivity hepatopathy and secondary liver cancer, and have two kinds of imaging examinations that the occupying lesion of liver cancer characteristic is arranged or have the positive and a kind of imaging examination of two kinds of liver cancer markers (DCP, GGT II, AFU and CA19-9 etc.) that the occupying lesion person of liver cancer characteristic is arranged.(c) clinical manifestation of liver cancer and the outer metastatic lesion (comprise macroscopic bloody ascites or find cancer cells therein) of sure liver is arranged and can get rid of the secondary liver cancer person is arranged.
Followed up a case by regular visits in the every 24-36 of patient month.Between each follow-up period, carry out liver function, AFP, hepatitis B virus s antigen (HBsAg), hepatitis B virus e antigen (HBeAg) detection.Follow up a case by regular visits to the preservation serum sample at every turn and be used for further analysis, carry out B ultrasonic simultaneously and detect.Clinical liver cirrhosis is defined as liver cirrhosis and hypersplenism sign feature.Follow up a case by regular visits to timing definition before the genesis of HCC for serum collection time first with diagnose out timed interval between the HCC.When following up a case by regular visits to when finish in June, 2009 every patient accessed 3-4 time.
Embodiment 2: experiment and statistical method
(Human 9-plex assay kit Utah) detects the human serum cytokine levels for Quansys Biosciences, Logan, and described test kit contains following 9 kinds of cytokines: IL-1 α to use Quansys, IL-1 β, IL-6, IL-8, IL-10, IL-17, IL-23, IFN γ and TNF α.Check and analysis are carried out in guidance according to manufacturer's handbook, and this analysis has high sensitivity and dynamics range (IL-1 α: 0.87-450pg/ml widely; IL-1 β: 3.24-1400pg/ml IL-6:3.81-475pg/ml; IL-8:0.21-125pg/ml; IL-10:0.22-190pg/ml; IL-17:1.63-450pg/ml; IL-23:18.64-8000pg/ml, TNF α: 0.73-350pg/ml; IFN γ: 0.25-100pg/ml exceeds the cytokine concentration of described dynamics range by computed in software).Each sample duplicate detection twice uses mean value to carry out data analysis.Whether influence cytokine stability in order to assess storage time length, in same people's serial blood sample, detected and the irrelevant people eosinophile cationic protein (ECP) of liver function.The result shows that ECP concentration does not change with the length in storage time, therefore thinks that the struvite cytokine that this experiment detected also is stable under suitable condition of storage.
During analytical data, continuous variable is represented with intermediate value and interquartile range.Detect the match stop variable by the side of card.Check patient who relatively develops into HCC and the cytokine levels between the contrast crowd who keeps chronic HBV infection by Mann-Whitney U.The time of following up a case by regular visits to of no HCC is by the Kaplan-Meier analysis and evaluation.The multiple logic regression model is used to adjust and the relevant latency of HCC generation.By the effect of experimenter's rational curve analysis and evaluation cytokine levels in prediction HCC takes place.All statistics tests are two tails.P＜0.05 thinks to have significance,statistical.
Embodiment 3: experimental result
To in June, 2009,45 patients develop into HCC, 45 ages and and the control patients that is complementary of sex matched.Developing into that median follow-up duration (interquartile range) is individual month of 108 (84,132) among the patient of HCC, is individual month of 144 (132,144) (p＜0.0001) in the control group.Some features (Table I) between we have also compared two groups.The The median age that develops into the patient of HCC is 45 (39,52) year, and control group is 44 (41,53) years (p=0.79).Because the male sex is more prone to develop HCC than the women, therefore in described group, 3 female patients are only arranged.28 (62%) patients that develop into HCC drink, and 29 (64%) patients drink in the control group.They have similar alcohol intake (p=0.72).In addition, also indifference of smoking state and family's medical history between two groups.
Table I-have and the do not have chronic viral hepatitis B patient's of HCC baseline characteristic
Figure BSA00000251213100121
We have detected 9 kinds of struvite cytokine spectrums.Develop into the patient of HCC and the cytokine-expressing spectrum of control patients and be shown in Table III.According to this table, we can find that IL-23 (a kind of important cytokine that plays a significant role) baseline values is developing between the case patient of HCC and the control patients and has significant difference in connecting innate immune responses and adaptive immune response.In the case patient, the median (interquartile range) of IL-23 (pg/ml) is 1315pg/ml (137-3816), apparently higher than the 227.9pg/ml (84.69-453.2) of control patients (interquartile range) (p=0.0014).
Experimenter's rational curve is used for also determining that IL-23 is in the developing predicting function of following HCC.The ROC area under a curve be 0.699 (95%CI 0.593/0.791) (Fig. 1).During positive likelihood ratio in selecting ROC curve each point/negative likelihood maximum, IL-23 concentration cutoff value is 855pg/ml.45 case patients and 45 control patients that we will develop into HCC are divided into two groups according to its baseline IL-23 concentration: high IL-23 group (＞855pg/ml) organize with low IL-23 (≤855pg/ml).In the case group, 27 (60.0%) patients have high IL-23 concentration, in control group, only have 4 patients to have high IL-23 concentration (p＜0.001), the susceptibility of prediction HCC development is 60.0%, and specificity is 91.1%, positive predictive value is 87.09%, and negative predictive value is 69.49%.
Table II-compare with control group, the ratio of IL-23 unconventionality expression significantly increases in the case group
Figure BSA00000251213100132
(1) susceptibility, the sample that promptly draws positive detection among the patient accounts for the per-cent TP/ (TP+FN) of patient's sum;
(2) specificity promptly draws the per-cent TN/ (FP+TN) that the negative sample that detects accounts for the control group sum in the control group;
(3) positive predictive value promptly draws in the total sample number of positive detection, and patient's sample accounts for the per-cent TP/ (TP+FP) of positive detection total sample number;
(4) negative predictive value promptly draws in the negative total sample number that detects, and check sample accounts for the negative per-cent TN/ (FN+TN) that detects total sample number.
The Kaplan-Meier analytical results shows that the CIR of high baseline IL-23 concentration and HCC is a significant correlation in the described research.144 25.42% 78.71% (p＜0.001) that increase in paramount IL-23 concentration patient of HCC CIR in the middle of the month in low IL-23 concentration patient.The logistic regression assay represent high IL-23 concentration (＞855pg/ml) be HCC development risk factor (for comprise age, sex, drink, the potential interfering factors of smoking and family history adjusts back OR=14.0295%CI 4.289/45.871p＜0.001).In last following up a case by regular visits to, the IL-23 level and the control patients that develop among the case patient of HCC do not have significant difference (p=0.599).We have also detected HCC and have diagnosed the change of preceding IL-23 concentration with respect to the time.Before research finished, we were divided into 5 time periods with sample.In control patients, IL-23 concentration keeps constant between follow-up period.In developing into the patient of HCC, higher in the early stage IL-23 concentration of progression of disease.Concentration reduces then, follows up a case by regular visits to indifference (Fig. 3) between the patient that developed into HCC before finishing in 0-24 month and the control patients.
Except IL-23, other 8 kinds of cytokine spectrums do not have significant difference at baseline values between case patient and control patients.
Figure BSA00000251213100151
Figure ISA00000251213300011

Claims (15)

  1. One kind identify the chronic HBV infection object for example the people have the method that the liver cancer tendency takes place, comprising:
    Detection derives from the level of IL-23 in the biological sample of described object,
    Wherein the rising of IL-23 level is that described object has the indication that the liver cancer tendency takes place in the test sample.
  2. 2. the process of claim 1 wherein that described biological sample is blood sample such as serum or derivatives thereof.
  3. 3. claim 1 or 2 method, the level of the mRNA of the level of IL-23 by detecting IL-23 albumen or IL-23 is determined in the wherein said sample.
  4. 4. each method of claim 1-3 wherein detects the proteic level of IL-23 in the described sample.
  5. 5. the method for claim 4 wherein detects the proteic level of IL-23 in the described biological sample by use IL-23 specific antibody or its Fab.
  6. 6. each method of claim 1-5, wherein in the test sample IL-23 level to raise be that the IL-23 level is compared with IL-23 level in the control sample and raise in the test sample.
  7. 7. claim 4 or 5 method, wherein in the test sample IL-23 level to raise be that the IL-23 protein level is higher than cutoff value in the test sample, for example be higher than 855pg/ml.
  8. 8. claim 4 or 5 method, wherein the proteic level of IL-23 is determined by detecting p19 subunit or its homologue in the sample.
  9. 9.IL-23 specific antibody or its Fab preparation be used for identifying the chronic HBV infection object for example the people have the application of the composition that the liver cancer tendency takes place.
  10. One kind be used to identify the chronic HBV infection object for example the people have the composition that the liver cancer tendency takes place, wherein said composition comprises the preparation of level that the biological sample IL-23 level that is used for detecting described object for example detects the mRNA of IL-23 albumen or IL-23.
  11. 11. the composition of claim 10, wherein said biological sample are for example serum or derivatives thereofs of blood sample.
  12. 12. the composition of claim 10, wherein said preparation are at the proteic specific antibody of IL-23, for example mono-clonal or polyclonal antibody.
  13. 13. be used to identify the chronic HBV infection object for example the people have the test kit that the liver cancer tendency takes place, described test kit comprises that the biological sample IL-23 level that is used for detecting described object for example detects the preparation of level of the mRNA of IL-23 albumen or IL-23.
  14. 14. the test kit of claim 13, wherein said biological sample are for example serum or derivatives thereofs of blood sample.
  15. 15. the test kit of claim 13 or 14, wherein said preparation are at the proteic specific antibody of IL-23, for example mono-clonal or polyclonal antibody.
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CN101221178A (en) * 2006-11-28 2008-07-16 创盛科技股份有限公司 Method for diagnosing primary malignancy hepatic tumor hepatocellular carcinoma
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WO2012025057A1 (en) * 2010-08-26 2012-03-01 中国医学科学院肿瘤研究所 Use of il-23 in early stage diagnosis of liver cancer
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