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Preparation, Identification and Effects on Proliferation of Human Hepatocellular Carcinoma Cell Lines of Monoclonal Antibodies Agaist LI-cadherin

Author: ChenZuoZuo
Tutor: WangSenMing；LiMing
School: Southern Medical University,
Course: Oncology
Keywords: Enterohepatic cadherin Monoclonal antibodies Hybridoma Tumor Appreciation
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 70
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Abstract

The research background HCC liver is the most common primary tumors, including hepatocellular carcinoma (heptocellular carcinoma, HCC) accounts for 90% of primary liver cancer in our country. The world die each year from the disease about a million people, the poor prognosis of advanced liver cancer, the overall 5-year survival rate is less than 5%. First, the degree of malignancy of primary liver cancer, endangering the majority of patients failed to early detection and early diagnosis and treatment when the majority belonging to the middle and late, lost the chance of operation, therefore, early detection, early diagnosis and early treatment is to improve the survival of patients with HCC and important prognostic factors, the increase in the rate of positive diagnosis of HCC patient survival and quality of life improvement is essential. The clinical application of alpha-fetoprotein (AFP) is mainly two things: first detection and monitoring of primary hepatocellular carcinoma, followed by monitoring treatment efficacy. AFP detection has been widely used for the diagnosis of primary liver cancer, but because of some liver cancer cells do not secrete this glycoprotein. Therefore can not be measured in these patients with primary liver cancer in vivo abnormal AFP. In some chronic liver disease, cirrhosis of the liver and gonads embryonal carcinoma, serum AFP moderate level of expression. So alone AFP diagnosis of primary liver cancer could easily lead to misdiagnosis and missed diagnosis, its clinical application is subject to certain restrictions. Secondly, metastasis and recurrence of the main factors also affect the liver cancer survival. Explore the development of liver cancer recurrence and metastasis related molecular mechanisms, and looking for new intervention targets liver cancer research has become a focus and difficulty. Interventional chemoembolization therapy and chemotherapy in the treatment of liver cancer, unresectable advanced hepatocellular carcinoma, hepatic artery is the main treatment, but either interventional therapy or chemotherapy, only a small portion of patients can benefit most patients poor efficacy, but also can not bring a survival benefit for patients. Such as doxorubicin traditional drugs for the treatment of liver cancer, but the remission rate was only 10% to 15%, and can not extend the survival time of patients. In recent years, with the continuous development of molecular biology techniques, new biological drugs continue to emerge, biological targeted therapy of patients with advanced hepatocellular carcinoma has gradually become a new choice, a new hope. This stage, molecular targeted therapy and as a basis for the radioimmunoassay targeted therapy made encouraging good efficacy in the treatment of advanced hepatocellular carcinoma, becoming the first choice for treatment of advanced HCC patients. Enterohepatic cadherin (CDH17) contains 832 amino acid residues, a molecular weight of about 92.3kD, belonging to a member of the cadherin protein family, but its structural features and biological behavior with traditional calcium fibronectin differ. CDH17 extracellular portion has seven structural domains, cell adhesion recognition area (cell adhesionrecognition CAR) the cadherin mediated specific adhesion, cytoplasmic portion contains 20 amino acid residues. Some studies have shown that CDH17 extracellular portion may act as a tool for the regulation of cell adhesion function alone. Under normal circumstances, CDH17 restrictive expression in rat liver and colon intestinal cells, never found CDH17 the presence and in the esophagus and stomach. Cadherin Ca ^{2 -dependent cell adhesion molecule, they played an important role in the occurrence and development of tissues and organs of the embryo formation and structure to maintain. If cadherin function disorder will be decreased so that the adhesion between cells, cell and tissue morphology changes, and thus give rise to the cell migration. This phenomenon will result in the invasion or metastasis of the outer periphery of the tumor cells in the tumor tissue. Therefore, cadherin can be regarded as one of the indicators of the patient's condition and prognosis tumor detection. Although CDH17 the structure is different from the traditional cadherin, but in the above-mentioned functions also play an equally important role. The CDH17 of dysfunction lead to the outer periphery of the tumor cell invasion and metastasis, eventually leading to a decline in tumor recurrence and overall survival, the results of a series of studies suggesting that CDH17 may become a diagnosis of cancer of the stomach, colon and liver cancer and / or prognostic indicators, monitoring progress and recurrence of the tumor. However, the specific role of CDH17 in tumor development process is not very clear, and provide the conditions for preparation of CDH17 monoclonal antibodies will CDH17 functional studies. In this study, purified recombinant CDH17 immunogen using the classic the lymphocyte fusion technology, the establishment of the hybridoma cell lines stably secreting specific monoclonal antibody purification and identification of monoclonal antibody, using Western-Blot the preparation of antibodies and immunohistochemistry experiments on the validation to reuse prepared anti-the CDH17 monoclonal antibody role in the human hepatoma HepG2 cells to investigate the role of its inhibition of liver cancer cell proliferation, and laid the foundation for further research CDH17 functionality. The purpose of preparation and identification of monoclonal antibody enterohepatic cadherin (CDH17), study of the hepatoma cell line HepG2 growth inhibition, to lay the foundation for the biological function of CDH17 protein. Method 1 animal immune selection of BALB / c mice with purified protein immune the indirect ELISA serum antibody titer of 1:6 400 or more. 2 cell fusion after the last immunization 3d take mouse spleen cells were fused with SP2 / 0 myeloma cells. CDH17 with purified recombinant fusion protein screening only CDH17 monoclonal antibodies reactive hybridoma cell line, line 1 Subcloning and 2-3 times a monoclonal hybridoma strain prepared and screened by indirect ELISA method. 4 induced ascites and antibody purified in one week in advance of injection of paraffin oil sensitized BALB / c mice were inoculated intraperitoneally built strains of hybridoma, collecting ascites, was purified by Protein-G affinity chromatography, SDS-PAGE analysis of purified results. 5 Western-Blot analysis to take with hepatoma cell line HepG2, QGY7703 Be17402, CBRH7919 on the samples to SDS-PAGE electrophoresis, an anti join anti-the CDH17 monoclonal antibody cell line culture supernatant F001 and F002 (dope). 6 Immunohistochemical analysis of data collected through the surgical removal of pathologically confirmed hepatocellular carcinoma tissue specimens, preparation of monoclonal antibodies for immunohistochemistry staining analysis CDH17 expression in hepatocellular carcinoma tissue. Control group with PBS instead of the first antibody. The 7 mAb role in HepG2 after cell morphology observed in logarithmic growth phase HepG2 cells were seeded in 6-well plates and cultured after 12 h with different concentrations of anti-CDH17 monoclonal antibody, its final concentration of 0 (control group, joined with volume medium), 5ug/ml, 10 ug / ml, 20 ug / ml, 40 ug / ml, 80 ug / ml, 24, 48, 72 and 96h after drug treatment under an inverted microscope continuous morphological changes were observed shooting and recording cell morphology. 8 MTT assay various concentrations of monoclonal antibody inhibition of tumor cell proliferation in logarithmic growth phase HepG2 cells were seeded in 96-well plates, and cultured 12 h with different concentrations of anti-the CDH17 monoclonal antibody, final concentrations of 0 (control group medium), 5ug/ml, 10 ug / ml, 20 ug / ml, 40 ug / ml, 80 ug / ml, were cultured 24, 48, 72 and 96h after the MTT assay concentration of monoclonal antibody to tumor inhibition of cell proliferation. 9 statistical processing experimental data obtained application SPSS15.0 statistical software for processing. Factorial design data variance analysis of the inhibition rate of the group and the time of each concentration group difference analysis, and SNK were its pairwise comparisons. P <0.05 for the difference was statistically significant. 2 Results Establishment of a hybridoma strain obtained a stable secretion of monoclonal antibody hybridoma strain, named F001 and F002. 2 McAb titer ELISA detected two monoclonal antibody (F001 and F002) of the cell culture supernatant antibody titer of 1:128 and 1:1024, ascites titer of 1:25600 and 1:51200. 3 antibody purity determination of two purified monoclonal antibody by SDS-PAGE electrophoresis, antibody purity of 80% or more. 4 Western blot results CDH17 in of HepG2, QGY7703, Be17402 and CBRH7919 in both the degree of expression of two monoclonal antibodies able to detect specific bands to the molecular size consistent with the CDH17 (92.3KD) forecast. Showed that CDH17 recombinant purified immunogen generated McAb can be combined with the natural eukaryotic cells CDH17 antigen. 5 immunohistochemical staining results of two anti-CDH17 monoclonal antibody with primary hepatocellular carcinoma tissue reaction can generate a brown or tan precipitation, mainly located in the cytoplasm of liver cancer cells, negative control, no brown or tan precipitate. 6 mAb role in HepG2 cell morphology changes 24, 48, 72 and 96h after dosing, control cells grew well, with the growth in the number of time increased with cells of uniform size, shape and was polygon, high-powered microscope cell chromatin loose, abundant cytoplasm. Cell culture experimental group 48 h after each experimental group were seen significantly reduced the number of cells, cell sizes, irregular shape, some cells became round, high-power microscopy showed nuclear condensation, cytoplasmic reduce and intracytoplasmic vacuoles, some cells off the wall was a semi-suspended state, time and concentration of the cell number and morphology changes and drug effects were positively correlated. 7 MTT assay various concentrations of monoclonal antibody in tumor cell proliferation inhibition by factorial variance analysis there are significant differences between drug concentration and the average inhibition rates between different time, and there was interaction between the two factors. Drug concentration and time factors from the inhibition rate of mutual promotion. Separate effects between different drug concentrations for the same duration of action, and the same concentration of the drug between the different time inhibition rates were significantly different, the same failure. Further SNK method to separate effects pairwise comparison results show that the addition 80ug/ml drug concentrations for 72h and 96h no significant differences exist between the different time between different drug concentrations for the same duration of action and the same drug concentration significant difference. Proved anti-CDH17 monoclonal antibody inhibits the proliferation of HepG2 cells with the increase of the concentration and prolonged duration of action, the inhibition of cell proliferation gradually increased, but when the the drug concentration 80ug/ml, for 72h or role 96h and There was no significant difference. Conclusions The use of hybridoma technology successfully screened and prepared two monoclonal anti-CDH17 antibody cell lines, and its specificity was identified by Western-blot and immunohistochemical identification, preparation of two monoclonal antibodies are and react specifically with human hepatoma cell lines and primary liver cancer pathology specimens, indicating that the prepared monoclonal antibody specific for CDH17. Further use of MTT assay anti-CDH17 monoclonal antibody on human hepatoma cell growth inhibition, indicating that the anti-human the CDH17 monoclonal antibody inhibits the proliferation of HepG2 cells in a time and concentration-dependent, with the increase of the concentration and prolonged duration of action, its inhibitory effect on cell proliferation gradually increased, and the synergy between time and dose.}

Preparation, Identification and Effects on Proliferation of Human Hepatocellular Carcinoma Cell Lines of Monoclonal Antibodies Agaist LI-cadherin

Abstract

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