CN106932587A

CN106932587A - Kit based on protein marker PSG3 auxiliary diagnosis liver cancer or alimentary tract cancer patient

Info

Publication number: CN106932587A
Application number: CN201511020625.5A
Authority: CN
Inventors: 肖汀; 程书钧; 陈实平; 高燕宁; 张开泰; 冯林; 郭宏林
Original assignee: Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2015-12-29
Filing date: 2015-12-29
Publication date: 2017-07-07
Anticipated expiration: 2035-12-29
Also published as: CN106932587B

Abstract

The kit based on protein label PSG3 auxiliary diagnosis cancers, especially liver cancer with colon cancer the invention discloses one kind.Application of the claimed product for detecting protein PSG3 in the kit for diagnosing liver cancer with colon cancer is prepared.In the present invention, inventor is found that the presence of protein marker PSG3 in liver cancer and peripheral blood of patients with colonic cancer first, and find that the concentration of protein marker PSG3 in liver cancer and peripheral blood of patients with colonic cancer has the value of auxiliary diagnosis liver cancer and colorectal cancer patients higher than healthy person, i.e. protein marker PSG3.The present invention is significant to the auxiliary diagnosis of liver cancer and colon cancer.

Description

Examination based on protein marker PSG3 auxiliary diagnosis liver cancer or alimentary tract cancer patient Agent box

Technical field

The present invention relates to a kind of applications of protein marker PSG3 in auxiliary diagnosis liver cancer or alimentary canal patient, and system Application in standby corresponding detection kit.

Background technology

Primary carcinoma of liver is the 6th, the whole world, Chinese 3rd most common cancer.According to IARC (IARC) Estimate, global onset of liver cancer number is 56.4 ten thousand people within 2000, wherein about 55% occurs in China, i.e., Chinese onset of liver cancer 30.6 ten thousand People, PLC mortality 30.0 ten thousand accounts for the dead second of China's Tumor of Residents.Belonging to middle and advanced stage when symptom occurs in liver cancer, after excision more Recurrence and metastasis rate is high.Therefore, the early diagnosis of liver cancer has important to the life span and reduction mortality of liver cancer that extend patient Meaning.

At present, imaging diagnosis, cell and histodiagnosis and chemical diagnosis is three big main methods of diagnosing tumor.Shadow Played an important role in diagnosing cancer of liver as learning diagnosis, but be respectively provided with diagnosis of small hepatic cell carcinoma and in distinguishing good Malignant Nodules certain Limitation.Ultrasonic examination or CT positive findingses, are higher than 400ng/ with reference to serum alpha-fetoprotein (α-fetoprotein, AFP) level The testing result of ml, can make to liver cancer and make a definite diagnosis.But, generally when these conditions all meet, had already passed by treatment most Good opportunity.Either ultrasonic examination, CT scan or nuclear magnetic resonance, have certain limitation, especially to the identification of small lesion It is the similarity that cirrhotic nodule has many on imageology with microhepatia cancerous node.Therefore, although Imaging Technology is obtained Huge progress, but clinically still need to combine the molecular marker of liver cancer, in complex case by good pernicious hepatopathy Make a distinction.Additionally, the clinical stages and tumor size of tumour are the determinants of liver cancer treatment effect, therefore, tumor-marker Thing can be additionally used in the generaI investigation of liver cancer population at risk (such as HBV chronic carriers and liver cirrhosis patient).

Current the most frequently used mark of diagnosing cancer of liver is alpha-fetoprotein (AFP), the number before symptom and Imageology occur The moon may occur in which exception.AFP contents are generally less than 10ng/ml in normal human serum.When the diagnostic value of AFP is set to 20ng/ml, Its sensitiveness is 50~60%, but in the less case of tumour, sensitiveness is significantly reduced, and has been reported that only 40%.Individually make Specificity is a lack of as another problem of diagnosing cancer of liver mark with AFP, in considerable chronic hepatitis patient especially In liver cirrhosis patient, AFP contents also reach 20-200ng/ml.It is now recognized that can have AFP heterogeneous with the mark of the complementary diagnosis of AFP Body, gamma glutamyltransferase isodynamic enzyme II and abnormal prothrombin etc., but due to they on Sensitivity and Specificity not Foot, and detection method very complicated, it is rested on laboratory research level all the time, fail to change into routine clinical inspection Project.Therefore, new tumor cells mark is explored, and sets up corresponding detection method easy to spread, be still current liver One of important topic of cancer research field.

Equally, tumor in digestive tract is also to cause one of Etiological of death in China.Colorectal cancer onset therein is hidden Hide, early stage is often without obvious clinical symptoms, and disease development is slower, patient occurs most to middle evening during manifest symptom Phase.At present, the treatment of colon is mainly based on operation, but chemotherapy effect after colon metastasis of cancer is poor, postoperative to there are about 40% There is recurrence in patient, and patients with terminal survival rates are relatively low.Do substantial amounts of on colon cancer in spite of many scholars The research of molecular level, but still lack the molecular marker for effectively early diagnosing colon cancer at present.Tumor markers can To be effective as clinical diagnosis foundation, people at highest risk, early diagnosis, guiding treatment, judgement treatment curative effect, detection can be monitored Recurrence and shift, be the important Index for examination of tumor patient, with important clinical meaning.The generation early stage of colon cancer has one The precancerous lesion of section time, such as polyp of colon, therefore in early detection and blocking or the development of delay cancer, there is extremely important Meaning.Therefore, find find valuable tumor markers for improve tumor in digestive tract, especially colorectal cancer diagnosis and Prediction occurs, development and improvement prognosis have important Clinical significance of MG.

Comprising abundant various cell components and molecular substance in human blood, can reflect well body different tissues and The physiology of organ, pathological state, and its sample is readily available, therefore, it is a kind of preferable tumour non-invasive diagnosis means.Therefore, Still there is the good mark of Sensitivity and Specificity to be developed.

Pregnant differential protein 3 (Human pregnancy specific beta-1-glycoprotein 3, PSG3), be One kind is secreted into extracellular glycoprotein.11 albumen are included in PSG families altogether, including PSG1, PSG2, PSG3 etc..PSG albumen It is general all to include the structure that N-terminal is similar to immune globulin variable region, it is followed by 2-3 constant region for immunoglobulin C2 spline structure Domain, and a short hydrophobic tail, positioned at C-terminal.PSG can be divided into 4 classes according to its structure composition, I types (N-A1-A2-B2-C), IIa types (N-A1-B2-C) and IIb types (N-A2-B2-C), type III (N-B2-C), IV types (A1-B2-C).Current PSG families Mechanism of action is unclear, under normal circumstances, great expression in the syncytionboph-oblast of placenta, outside gestational period women PSG protein contents are raised in all blood, up to 200-400 μ g/ml.It is rarely reported on research of the PSG families in cancer.

428 amino acid of PSG3 length proteins, molecular weight 47,945Da, following (the SEQ ID of amino acid sequence of albumen NO：1)：

MGPLSAPPCT QRITWKGLLL TALLLNFWNL PTTAQVTIEA

EPTKVSKGKD VLLLVHNLPQ NLAGYIWYKG QMKDLYHYIT

SYVVDGQIII YGPAYSGRET VYSNASLLIQ NVTREDAGSY

TLHIVKRGDG TRGETGHFTF TLYLETPKPS ISSSNLYPRE

DMEAVSLTCD PETPDASYLW WMNGQSLPMT HSLQLSKNKR

TLFLFGVTKY TAGPYECEIR NPVSASRSDP VTLNLLPKLP

KPYITINNLN PRENKDVLAF TCEPKSENYT YIWWLNGQSL

PVSPRVKRPI ENRILILPSV TRNETGPYQC EIQDRYGGIR

SYPVTLNVLY GPDLPRIYPS FTYYHSGENL YLSCFADSNP

PAEYSWTING KFQLSGQKLF IPQITTKHSG LYACSVRNSA

TGMESSKSMT VKVSAPSGTG HLPGLNPL

Research report about PSGs in tumour is less.The application differential gene expression analysis of spectrum such as Salahshor finds Abnormal elevated PSG9 transcripts are can detect in colorectal cancer, metastatic liver cancer, cervix tumor tissue, it is viscous with normal Colon and rectum Film is compared, and PSG9 raises 2 times in adenoma, and colorectal cancer PSG9 abnormal expressions are raised and are mutated dependence in APC (Salahshor et al,2005).The research of Kamarli ZP etc. shows, PSG1 albumen in bone malignant tumour patients serum Concentration raises (Kamarli et al, 2004).And the research on PSG3 in tumour has not been reported.

The content of the invention

First aspect present invention is related to the molecule for detecting PSG3 albumen preparing for auxiliary diagnosis cancer, especially liver cancer Or the purposes in the kit or detection reagent of alimentary tract cancer patient.

The purposes of any one according to a first aspect of the present invention, wherein the molecule of the detection PSG3 albumen be refer to it is special Property detection PSG3 protein expressions molecule, for example can be nucleic acid, protein or compound.

The purposes of any one according to a first aspect of the present invention, wherein the protein is antibody, it can be with PSG3 albumen Specific binding, preferably monoclonal antibody.

In embodiments of the invention, the molecule of the detection PSG3 albumen is monoclonal antibody, and it can be with PSG3 Protein-specific is combined.

In specific embodiments of the present invention, PSG3 albumen is detected using immunochemical method.

The purposes of any one according to a first aspect of the present invention, wherein described liver cancer is hepatocellular carcinoma.

The purposes of any one according to a first aspect of the present invention, wherein described alimentary tract cancer is colorectal cancer.

The purposes of any one according to a first aspect of the present invention, the kit or detection reagent are used for the inspection of plasma sample Survey.

The purposes of any one according to a first aspect of the present invention, wherein the determination methods of the auxiliary diagnosis are, in blood plasma The content of PSG3 is higher than 551.65 μ g/ml, then person to be checked is the hepatocellular carcinoma or colorectal cancer patients of candidate.

The purposes of any one according to a first aspect of the present invention, wherein the molecule of the detection PSG3 albumen is also with detectable Mark.

The purposes of any one according to a first aspect of the present invention, wherein also containing buffer solution in the kit or detection reagent And developer.

It is related to a kind of examination for auxiliary diagnosis cancer, especially liver cancer or alimentary tract cancer according to a second aspect of the present invention Agent box or detection reagent, its product for containing detection label PSG3 albumen.

It is related to a kind of examination for auxiliary diagnosis cancer, especially liver cancer or alimentary tract cancer according to a second aspect of the present invention Agent box or detection reagent, the product of the detection label PSG3 albumen are to refer to dividing for specific detection PSG3 protein expressions Son, for example, can be nucleic acid, protein or compound.

The kit or detection reagent of any one according to a second aspect of the present invention, wherein the protein is antibody, its energy Enough combined with PSG3 protein-specifics, preferably monoclonal antibody.

The kit or detection reagent of any one according to a second aspect of the present invention, wherein described liver cancer is hepatocellular carcinoma, Described alimentary tract cancer is colorectal cancer.

The kit or detection reagent of any one according to a second aspect of the present invention, the kit or detection reagent are used for blood The detection of slurry samples, it is preferable that the detection method is immuno-chemical method.

The kit or detection reagent of any one according to a second aspect of the present invention, wherein the determination methods of the auxiliary diagnosis For blood plasma PSG3 protein expressions are higher than 551.65 μ g/ml, then person to be checked is the hepatocellular carcinoma or colorectal cancer patients of candidate.

The kit or detection reagent of any one according to a second aspect of the present invention, wherein the molecule of the detection PSG3 albumen Also carry detectable mark.

The kit or detection reagent of any one according to a second aspect of the present invention, wherein in the kit or detection reagent Also contain buffer solution and developer.

In the present invention, wherein the molecule of the detection PSG3 albumen refer to detection PSG3 albumen whether overexpression Molecule, for example, can be nucleic acid, protein or compound.

In the present invention, antibody can be prepared using method well known in the art.

The method of immunoassays can be used quantitative or qualitative detection protein marker presence.The immunoassays are usual Including biological sample is incubated together with antibody, and know technology for detection binding antibody by various.

In the present invention, the compound has implication well known in the art, and it can be combined with PSG3 protein-specifics, And then for detecting the expression of PSG3 albumen.

In the present invention, detectable mark molecule can be connected with the molecule of the detection PSG3 albumen.

In the present invention, can also be included in the kit or detection reagent can be with the molecule knot of detection PSG3 albumen The molecule of conjunction, such as antibody, such as SA, the molecule can also be connected with detectable mark molecule.

In the present invention, the detectable mark molecule for example can be enzyme, fluorescein, metal ion or isotope Deng.

Brief description of the drawings

Figure 1A is the ROC curve that PSG3 distinguishes normal person and patients with hepatocellular carcinoma (HCC).

Figure 1B is the ROC curve that PSG3 distinguishes normal person and colorectal cancer patients (CC).

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can by city available from conventional products.

Embodiment 1 prepares the ELISA kit of PSG3 detections

Using escherichia coli prokaryotic expression PSG3 albumen, expression vector is pET30a.5-6 week old BALB/c mouses are taken, profit With PSG3 full-length proteins the immune preparation PSG3 antibody of Freund's adjuvant is added as antigen.Every mouse immune 4 times, per minor tick 2 In week, 15-30 μ g antigen proteins are utilized every time, Freund's complete adjuvant is utilized during first time inoculation, utilize Freund's incomplete adjuvant three times afterwards. First three day of cell fusion, selecting one from three spleen cell donor mices carries out intravenous injection PSG3 antigens and does further to be immunized. Cell fusion, hybridoma screening are according to the method (Davis et al 1982) of J.M.Davis.Simple process is as follows：Strengthen Mouse boosting cell and SP2/0 myeloma cell mixing after immune, is slowly added into rush fusion agent 50% polyethylene glycol 0.7ml.37 DEG C culture 1min, 10ml IMDM culture mediums dilution, low-speed centrifugal.Then 1.25% methylcellulose, 25% tire ox are contained with 40ml Serum, 2%HAT (contain hypoxanthine, methotrexate (MTX) and thymidine) IMDM culture mediums it is resuspended.Turn upside down mixing, It is transferred in 35mm flat boards, per plate about 2ml, in 37 DEG C, 5%CO₂Cultivated in environment.7 days rear clones are transferred to 96 orifice plates, add IMDM culture mediums containing 15% hyclone and 2%HT (containing hypoxanthine and thymidine) are cultivated again.When cell melts It is right to reach 50%~70%, culture medium supernatant is collected, detect whether there is antibody using ELISA and Western blot.

Set up the enzyme mark double antibody sandwich method of detection PSG3 antigens.Detect that the enzyme mark double antibody sandwich method of PSG3 antigens is built It is vertical：Monoclonal antibody is produced first with mouse peritoneal, the positive hybridoma cell that will be obtained is injected separately into mouse peritoneal, makes it Ascites is produced, norphytane, every mouse 0.5ml is injected；Injection norphytane injects hybridoma, every mouse injection after 1 week 10⁶Individual cell.Ascites is collected after 7-10 days respectively, antibody is purified respectively with caprylic acid-ammonium sulfate precipitation method, then use sodium periodate Antibody is carried out method horseradish peroxidase-labeled (enzyme labelled antibody) respectively.Enzyme mark is coated with respectively with the monoclonal antibody of purifying Plate, and PSG3 antigens are detected with enzyme labelled antibody pairing respectively, the coated antibody P201 of acquisition best pairing and the capture of enzyme mark are anti- Body P209.Wherein coated antibody P201 and enzyme target capture antibody P209 are prepared by hybridoma cell strain P201 and P209 produce respectively It is raw.Hybridoma cell line P201 and P209 are common in China Committee for Culture Collection of Microorganisms on November 27th, 2015 Microorganism center preservation, preserving number is respectively CGMCC NO.11597 and CGMCCNO.11598, and preservation address is exposed to the sun for Beijing The institute 3 of area North Star West Road one.

ELISA steps are as follows：By PSG3 coated antibodies P201 96 hole elisa Plates of coating, 100 μ l/ holes, 4 DEG C overnight；Get rid of Coated antibody, 0.5%PBST board-washings 3 times,2%BSA confining liquids are added,Room temperature 4 hours；Get rid of Confining liquid, 0.5%PBST board-washings 3 times,PSG3 standard proteins and sample to be tested are separately added into per hole in ELISA Plate, 100 μ l/ holes；Add PSG3 enzyme labelled antibodies P209,100 μ l/ holes, 4 DEG C overnight after mixing；Get rid of dereaction liquid, 0.5%PBST Board-washing 5 times, TMB nitrite ions are added, 200 μ l/ holes, lucifuge is reacted 15 minutes；2M sulfuric acid terminating reactions are added,With detecting absorbance at ELIASA OD450nm；Standard curve is drawn using the value of standard protein absorbance, and is counted Calculate the protein concentration of PSG3 in sample to be tested.

Embodiment 2 detects the protein concentration of PSG3 in large-scale tumor patient and normal population plasma sample

The concentration of PSG3 albumen is detected in pregnant woman's blood plasma using the ELISA kit for preparing.The sample bag of detection Include：11 early pregnancy women (11-12 weeks), 17 Advanced pregnant women (28-38 weeks).The result of ELISA as shown in Table 1, Protein concentrations of the PSG3 in early pregnancy women's blood plasma is significantly higher than Advanced pregnant (P<0.001).

Protein concentrations (μ g/ml) of the table 1.PSG3 in pregnant woman's blood plasma

Detect PSG3's in large-scale tumor patient and normal population plasma sample using the ELISA kit for preparing Protein concentration.The sample of detection includes：178 hepatocellular carcinomas (Hepatocarcinoma, HCC), 121 lung cancer (Lung Cancer, LC), 159 oophoromas (Ovarian Cancer, OC), 88 colorectal cancers (Colorectal Cancer, CC), And the healthy normal person (Health Control) that 115 ages match with sex ratio.The result of ELISA shows, PSG3 Normal group (P is significantly higher than in the plasma protein levels of hepatocellular carcinoma, colorectal cancer patients and patients with lung cancer<0.001, Table 2).

Protein concentrations (μ g/ml) of the table 2.PSG3 in each group tumor patient and normal population blood plasma

Effect of the PSG3 plasma protein concentrations of embodiment 3 in auxiliary diagnosis of hepatoma or alimentary tract cancer

When PSG3 plasma protein concentrations value is 551.65 μ g/ml, the sensitiveness for detecting hepatocellular carcinoma is 64.2%, special The opposite sex is 74.8%, and area is 0.774,95%CI=0.719-0.828, P under ROC curve<0.001 (Figure 1A).Same group of liver Also the level of hepatocellular carcinoma mark alpha-fetoprotein (alpha-fetal protein, AFP) is have detected in carninomatosis human plasma. When the use of routine clinical detection limit value being 25ng/ml, the patient of hepatocellular carcinoma AFP negative is 107, accounts for total specimens 60.1% (107/178).And in this 107 hepatocarcinoma patients of AFP negative, positive (the PSG3 protein concentrations in blood plasma of PSG3> 551.65 μ g/ml) case load be 66, improve the recall rate of hepatocarcinoma patient, can clinically aid in hepatocellular carcinoma disease The diagnosis of people.

When PSG3 plasma protein concentrations value is 551.65 μ g/ml, the sensitiveness of detection colorectal cancer (n=88) is 92.0%, specificity is 74.8%, and area is 0.873,95%CI=0.819-0.828, P under ROC curve<0.001 (Figure 1B). This 88 patient's blood plasma also have detected colorectal cancer mark carcinomebryonic antigen (carcinoembryonic antigen, CEA) Protein level.When the use of routine clinical detection limit value being 5ng/ml, CEA negative patient is 67 in colorectal cancer patients, Account for the 76.1% (67/88) of total specimens.And in the negative Patients with Colorectal Cancer of this 67 CEA, the PSG3 positives are (in blood plasma PSG3 protein concentrations>551.65 μ g/ml) case load be 60, significantly improve the recall rate of Patients with Colorectal Cancer, it is clinical On have preferable application prospect.

Bibliography

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Claims

1. it is used to detect application of the product of protein marker PSG3 in the kit for auxiliary diagnosis cancer patient is prepared.

2. the application of claim 1, wherein the cancer is liver cancer or alimentary tract cancer.

3. the kit or detection reagent of auxiliary diagnosis cancer patient are used for, wherein the product containing detection protein marker PSG3 Product.

4. the kit or detection reagent of claim 3, wherein the cancer is liver cancer or alimentary tract cancer.

5. the application of claim 2 or the kit of claim 4 or detection reagent, wherein the liver cancer is hepatocellular carcinoma, institute The alimentary tract cancer stated is colorectal cancer.

6. the application described in foregoing any one claim or kit or detection reagent, the kit or detection reagent are used for The detection of plasma sample, it is preferable that the detection method is immuno-chemical method.

7. the application described in foregoing any one claim or kit or detection reagent, wherein detection protein marker PSG3 Product is the molecule for referring to specific detection PSG3 protein expressions, for example, can be nucleic acid, protein or compound.

8. application described in claim 7 or kit or detection reagent, wherein the specific detection PSG3 protein expressions Molecule is antibody, preferably monoclonal antibody.

9. the purposes or kit or detection reagent described in any one of claim 7 or 8, wherein the detection PSG3 protein expressions Molecule also carry detectable mark.

10. the purposes or kit or detection reagent described in foregoing any one claim, wherein the kit or detection examination Also contain buffer solution and developer in agent.