| BackgroundLiver cancer is the most common primary liver cancer,one of hepatocellular carcinoma(heptocellular carcinoma,HCC) in China accounted for 90%of primary liver cancer.Each year about one million people died of the disease all over the world,liver cancer and poor prognosis,the survival rate of overall 5-year is less than 5%.First of all,the primary malignant liver cancer is of high hazard degree,and the majority of patients are failed in early detection and early diagnosis.When seek medical treatment,the majority belong to the late period and lose the surgery opportunity,therefore,early detection,early diagnosis and early treatment are important factors of improving the survival of HCC patients and prognosis,The increase of the positive rate of diagnosis is essential for improving the positive rate of HCC patients and survival quality of life.There are two main clinical application of Alpha-fetoprotein(AFP):First of all detect and monitor primary hepatocellular carcinoma,followed by monitoring the therapeutic effect.Although the AFP has been widely used in detection of primary liver cancer diagnosis,but because of certain hepatoma cells do not secrete this glycoprotein.Therefore,in some patients with primary liver cancer can not be measured in vivo abnormal AFP.However,in certain chronic liver disease,cirrhosis and carcinoma embryonic gonad,the serum AFP level has the expression in middle degree.Therefore,it is easy to missed diagnosis and misdiagnosis when diagnose primary liver cancer by AFP alone, so its clinical application are subject to certain restrictions.Secondly,metastasis and recurrence are also major factors for affecting the survival rate of liver cancer.To investigate the occurrence and development of liver cancer,metastasis and recurrence-related molecular mechanisms,and find new targets for intervention have become the focus and difficult point of the study of liver cancer.In treatment of liver cancer,for advanced hepatocellular carcinoma which cannot be cut by surgical resection,transcatheter arterial chemoembolization therapy and chemotherapy are the most important means of treatment.No matter the intervention or chemotherapy,only some patients can receive benefit.The effect of majority patients was not obvious,and it could not bring about the survival benefit for patients. Currently,doxorubicin is a traditional drug for treatment of liver cancer,but the remission rate was only 10%～15%,and it could not extend the survival time of patients.In recent years,with the development of molecular biology techniques,new biological drugs continue to emerge,biological targeted therapy has become a new choice,new hope for advanced liver cancer patients.At this stage,molecular targeted therapy and the RIA-based targeted therapy have achieved encouraging good efficacy in the treatment of advanced liver cancer and have become the first choice for treatment for patients with advanced HCC.Liver cadherin contains 832 amino acid residues,molecular weight is about 92.3kD,belonging to cadherin family members,but its structural features and biological behavior are different from traditional cadherin.Extracellular part of CDH17 have 7 structure regions,cell adhesion recognition region(cell adhesion recognition CAR)-mediated specificity of cadherin adhesion,cytoplasmic part contains 20 amino acid residues.Research has shown that CDH17 extracellular part maybe a tool that is solely regulating cell adhesion.Under normal circumstances,CDH17 restrictively exists in rat liver,small and large intestine cells, but never found in esophagus and stomach.Cadherin is a cell adhesion molecule on which Ca2 + depends.They play an important role in embryo development and the formation of organs and structures to maintain.The dysfunction of cadherin leads to the decline of cell adhesion,morphological changes of cells and tissues,thus cell will be migration.This phenomenon appears in the tumor tissue will lead to tumor cell infiltration or transfer on the outward.Therefore,cadherin can be considered as one of the guidelines of patients condition and prognosis.Although the CDH17 structure and the traditional cadherin are different,but they played an equally important role in the above-mentioned features.Dysfunction of CDH17 will lead to tumor cell invasion and metastasis on the outward,eventually leading to tumor recurrence and decline of overall survival rate.A series of studies suggest that CDH17 may become guideline of tumor diagnosis and/or prognostic of stomach,colon and liver cancers,etc and monitor the tumor recurrence and progress.However,the concrete function of CDH17 is not very clear in tumor occurrence and development,monoclonal antibodies of CDH17 preparation will provide condition for the function research.In this research,purified recombinant CDH17 is used as immunogen,using the classic lymph cell fusion technique to set up a stable secretion of specific monoclonal antibody hybridoma cell line,and purified and characterized the obtained monoclonal antibody.Besides,the antibodies is verified by Western-Blot and immunohistochemical experiments and then use prepared the anti-CDH17 monoclonal antibody acting on human hepatoma HepG2 cells to explore its inhibition of liver cancer cell proliferation,so that it lays the foundation for the further study of the function of CDH17.PurposePrepare and identify monoclonal antibody of liver cadherin(CDH17) in order to study their inhibitive impact on hepatocellular carcinoma cell line HepG2 growth and then lay the foundation of the investigation in the biological function of protein CDH17.Methods1.Animal immunity.Choose BALB/c mice and purified immunity,indirectly use ELISA to test serum antibody titer reached 1:6 400 or more.2 Cell fusion.After the last immunization,we use 3d to take spleen cells from mice with SP2/0 myeloma cell to fusion.3 Preparation and screening of hybridoma cell line and screening.Using indirect ELISA method with purified recombinant CDH17 fusion protein only with CDH17 reaction of the monoclonal antibody hybridoma cell line and one time of subcloning and 2-3 times of monoclonal.4.induced ascites and purification of antibodies.Inoculate hybridoma cell lines that have been built in the abdomen of the BALB/c mouse which is sensitized by the injection of paraffin oil before 1 week.Collecting ascites and purified by Protein-G affinity chromatography and using SDS-PAGE to analyse the result.5 Western-Blot analysis.Choose separately with hepatocellular carcinoma cell line HepG2,QGY7703,Bel7402,CBRH7919 on samples to SDS-PAGE electrophoresis,one anti add anti-1 anti-CDH17 monoclonal antibody cell lines culture supernatant F001 and F002(stock solution).6 Immunohistochemical analysis.Collect tissue samples that are confirmed primary hepatocellular carcinoma by the pathology after surgical resection.Use the prepared monoclonal antibodies for immunohistochemical staining to analyze CDH17 in hepatocellular carcinoma tissues.PBS in place of the first antibody in comparison group7 Observation of monoclonal antibody acts on HepG2 cell morphology.After 12 h culturing of logarithmic grow phase HepG2 cells inoculated in 6-well plates,adding different concentrations of anti-CDH17 monoclonal antibody,and its final concentration are 0(add the same volume of medium in comparison group),5ug/ml, 10 ug/ml,20 ug/ml,40 ug/ml,80 ug/ml.After drug treatment 24,48,72 and 96h,continuously observe cell morphological changes under the inverted microscope and record the cell morphology.8 Assay the inhibition of concentration of monoclonal antibody on tumor cell proliferation by MTT.Logarithmic phase HepG2 cells inoculated in 96-well plates, adding different concentrations ofanti-CDH17 monoclonal antibody after cultured 12 h,and its final concentration are 0(add culture medium in comparison group,), 5ug/ml,10 ug/ml,20 ug/ml,40 ug/ml,80 ug/ml,.After respectively culture 24,48,72 and 96h,using MTT to assay the inhibition of concentration of monoclonal antibody on tumor cell proliferation.9 Deal with statistics.Data from experiment should use the SPSS15.0 statistical software to deal.Use homogeneity of variance test to confirm their homogeneity.etc to satisfy conditions to the information factorial design analysis of variance for difference analysis,using SNK method comparison between the twos.P＜0.05 for differences with statistical significance.Results1 Establishment of hybridoma cell line.To obtain two strains of stable monoclonal antibody secreting hybridoma cell line, named for the F001 and F002.2 McAb titer Detecte.Two monoclonal antibodies(F001 and F002) of the cell culture supernatant antibody titer to 1:128 and 1:1024,ascites titer to 1:25600 and 1:51200 by ELISA.3 Determination of antibody purity.After purification of two monoclonal antibodies by SDS-PAGE electrophoresis, purity of antibodies has more than 80%.4 results of Western blot.The results showed that CDH17 has expression in varied degrees in HepG2, QGY7703,Bel7402 and CBRH7919.the two monoclonal antibodies can be detected with the CDH17(about 92.3KD) the size of the same molecular weight prediction specific band.It is showed that purified recombinant CDH17 for the immunogen produced McAb can integrate with eukaryotic cells within the natural antigen-binding CDH17.5 results of immunohistochemical staining.Two Strains of CDH17 monoclonal antibody and anti-hepatocellular carcinoma tissue reaction can generate brown or tan-precipitation,mainly located in the cytoplasm of hepatoma cells,negative control,no brown or brown precipitate. 6 morphological changes of monoclonal antibody acting on the HepG2 cells.After dosing 24,48,72 and 96h,the control group cells grew well,with growth of quantity of time and an increase in growth,cell size are uniform,with same shape and in line polygon,The cells loose chromatin,abundant cytoplasm can be seen under high-power microscope.After 48h of cell cultured of experimental group,the experimental group were significantly reduced the number of visible cells,cell sizes, shape irregular,some cells became round,high-power microscopy shows that nuclear pyknosis,reduced cytoplasmic vacuoles and the cytoplasm appeared,some cell degranulation was a half wall in suspension,cell number and morphological changes with time and drug concentration was positively correlated.7 Assay the concentration of monoclonal antibody on inhibition of tumor cell proliferation by MTT.Anti-CDH17 monoclonal antibody on HepG2 cell proliferation inhibitory effect was time-and concentration-dependent,with the concentration and the role of the increase in time,its inhibitive function on cells increase gradually.After 96h the role of HepG2 cell proliferation the largest inhibition rate was(29.4±0.5)%.By analysis of variance,it is not very the same between different drug concentration and the different roles between the time the average inhibition rate and the two interaction effects between factors.Note the role of drug concentration and time factors on the inhibitory rate of the two promote each other starting role.Further using SNK method to compare results between the twos showed that the concentration between the different drugs and different time are different between the subgroups.Further evidence of anti-CDH17 monoclonal antibody on HepG2 cells with increasing concentrations and the role of time,its inhibitive function on cells increase gradually.ConclusionSuccessfully established two titer and good specificity of anti-CDH17 protein monoclonal antibody,which can inhibit HepG2 cell proliferation in a dose-time -dependent effect. |
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