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Preparation and Identification of Monoclonal Antibody Against Human Stathmin and Inhibitory Effect of the McAb on Hepatoma Carcinoma Cells

Author: YuanShaoZuo
Tutor: WangSenMing；LiMing
School: Southern Medical University,
Course: Oncology
Keywords: stathmin Monoclonal antibodies Taxol Hepatic carcinoma
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 184
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Abstract

Primary liver cancer (Primary Hepatic Carcinoma, PHC) is the world's most common and one of the most malignant tumors, the incidence of malignant tumors in five of the highest in the world, the mortality rate among the three. China due to the presence of a large number of chronic hepatitis B infection crowd of primary liver cancer is still of a high incidence of major diseases, an incidence of 30.3 per 100 million each year about 14 million people died of PHC cent of the world's PHC death more than 50% of the number of persons. Liver cancer due to occult onset, rapid progress, and clinical outcomes remains poor. Currently, the clinical diagnosis of liver cancer is not difficult, but the majority of cases are in the late clinical findings. For the treatment of liver cancer include surgical treatment of hepatic artery / portal vein chemoembolization, ablation and radiation therapy have a certain effect, using a variety of treatments to comprehensive treatment of liver cancer patients liver cancer treatment trends. The late 20th century, people began to use a systems biology perspective to study the tumors, recognize tumor is a multifactorial participate in the development of multi-step generalized systemic disease. Caused by mutations of oncogenes, tumor suppressor gene is missing or disorders caused by the tumor genome stability loss in regulation of gene expression, leading to the biological behavior of the tumor cells appear abnormal. Molecular targeted therapy refers to the use of small molecules, monoclonal antibodies, peptides and other substances specifically interfere with the signaling pathways that regulate the biological behavior of tumor cells, thereby inhibiting tumor growth. Molecular targeted therapy essentially different from traditional chemotherapy, molecular targeted therapy has a high selectivity for tumor abnormal signaling pathways, low toxicity and high therapeutic index, long-term medication. Cell division, the chromatids spindle on the movement depends on the dynamic instability of microtubules, microtubules regulate the expression of the disorder can disrupt mitosis, abort the growth of malignant cells. Stathmin is a microtubule destabilizing protein, a newly discovered play a very important role in regulation of cell microtubule system power balance in the different stages of the cell cycle by phosphorylation and dephosphorylation. Variety of malignant tumors of stathmin are expressed at high levels by inhibiting its expression can interfere with the malignant cell division. Studies have shown that, stathmin provide new molecular targets for gene therapy in a tumor. The Stathmin are filtered out from the lymphoma characteristic gene expression product, consisting of 149 amino acids, the molecular weight of about 19kD, widespread in the vertebrate cells, the highly conserved cytoplasmic phosphoprotein. Signal transduction studies have shown: the cell, the outer product of a variety of cytokines, oncogenes and tumor suppressor gene expression directly or indirectly caused by changes in cell biology and stathmin role. The Stathmin multiple intracellular kinase substrates, and its downstream targets of play a key role in the cell division cycle tubulin and microtubules and spindle organelles. Regulate microtubule dynamics balance, stathmin can control the cell cycle, and change the cell proliferation, differentiation and activity of biological behavior. Stathmin promote micro-tube depolymerization of activity by its own phosphorylation levels of adjustment with respect to the cell interphase and mitosis stathmin phosphorylation levels significantly with increased, its amino end of the four serine by cell cycle factors dependent kinase, have silk mitogen-activated protein kinase, calmodulin-dependent kinase and cAMP-dependent protein kinase kinase role of phosphorylation. The study confirmed that the induction of wild-type stathmin will lead to over-expression of cell interphase microtubule depolymerization. When these cells enter mitosis early, stathmin is inactivated by phosphorylation, so that the tubulin polymerization and spindle assembly. Mitosis late stathmin dephosphorylation of mitosis is required for the disintegration of the spindle and exit. Kinase target mutated stathmin expression of the mutant, the cells can not make stathmin phosphorylation, microtubule not capable of polymerization and the formation of a functional spindle. These findings suggest that stathmin phosphorylation is critical to inactivation of the spindle formation and cell cycle progresses. The Stathmin the high levels of expression can be leukemia, lymphoma, prostate cancer, ovarian cancer, lung cancer, liver cancer, into a detected in the osteosarcoma, breast cancer and other tumors. Alli and other found to contain mutant p53 gene stathmin expression in human breast cancer cell lines. Mistry further research experimental tumor models in cancer-causing gene-mediated transformation of stathmin expression levels, suggesting enhanced tumor cell proliferation activities only a reflection of the high level of expression of the stathmin often observed in tumor cells. Subsequent studies have confirmed that the high level expression of stathmin the maintenance of tumor cells in high proliferation rate is required. The drugs inhibit cell division by interfering with the microtubule function has been widely used in cancer chemotherapy, paclitaxel and vinblastine two chemotherapy drugs. The study confirmed that the of stathmin overexpression and lung cancer cells of Changchun ground Xinmin flu-related, can be used as a sensitive sign of lung cancer patients Changchun alkaloids. Balachandran confirmed paclitaxel (Taxol) and anti-stathmin ribozyme combination therapy can provide a more efficient, low toxicity of cancer treatment, thus greatly reducing the variety of chemotherapy drugs in combination lead to overlapping cumulative toxicity. The stathmin protein an important role in the spindle regulation and cell transformation, stathmin gene and its expression product is a highly attractive malignant biological treatment of new target, stathmin has become an attractive molecular cancer gene therapy goals. In recent years, stathmin gene therapy experiment continued success. Stathmin essentially all transformed cells have a high level of expression, stathmin can be a very good biological therapeutic targets for the treatment of tumors, the role of microtubule chemotherapy drug paclitaxel combination has a broad prospect. Therefore, this study of purified recombinant stathmin protein as an immunogen, using classic Lymphocyte fusion technology, establish stably secrete monoclonal antibodies specific hybridoma cells, the monoclonal antibodies obtained were purified and identified. antibody prepared using Western-Blot and immunohistochemistry, and further study of anti-stathmin monoclonal antibody combined with paclitaxel on human hepatoma HepG2 cell growth inhibition, has laid for the the stathmin protein function further research and clinical applications foundation. Objective To prepare microtubules unstable protein (stathmin) monoclonal antibody to identify its specificity, and to explore the anti-human stathmin monoclonal antibody, paclitaxel alone and in combination with the hepatoma cell line HepG2 growth inhibition. Method 1. Material of BALB / c mice, female, 6 to 8 weeks of age, about 20g, were purchased from the Experimental Animal Center of Sun Yat-sen University. SP2 / 0 myeloma cells, the hepatoma cells the strains QGY7703 and HepG2, Southern Medical University, Institute of antibody engineering laboratory passaged save. The stathmin protein as immunogen purification Southern Medical University, Institute of antibody engineering. 2 experimental step experiments conducted in two parts: 2.1 anti-human stathmin monoclonal antibodies and identification 2.1.1 animal immune selection of BALB / c mice, female, 6 to 8 weeks old, 20g. Immunization with the purified protein, by indirect ELISA serum antibody titer of 1:6 400. 2.1.2 cell fusion of mouse spleen cells were fused with SP2 / 0 myeloma cells, 50% PEG solution for the integration of agent fusion cells with HAT selective medium, after the switch to the first 7d HT medium first 17d after change Ordinary RPMI-1640 culture medium. 2.1.3 induced ascites and purified by antibody in one week in advance of injection of paraffin oil sensitized BALB / c mice were inoculated intraperitoneally with strains of hybridoma has been built, 10d collected after ascites and centrifuged, the supernatant, the ascites supernatant filtration after dilution, was purified by Protein-G affinity chromatography column, and desalted using SephadexG50 column, and collecting the protein peak purified by SDS-PAGE analysis results. 2.1.4 Western-Blot analysis of cultured human hepatoma cell lines HepG2 and QGY7703, take the 1 × 10 ^{7 cells frozen for 2min, remove the added 600μl 1 × SDS sample buffer, boiling water for 5min , 10000r/min centrifugal 5min. Press sample sequentially, were added to a different antibody, was further added to the secondary antibody (HRP-labeled goat anti-mouse IgG polyclonal antibody). Adding chemiluminescent substrate reaction solution processing, into the X-ray photography of the cartridge, and the developing and fixing in the darkroom. 2.1.5 Immunohistochemical analysis of pathologically confirmed primary liver cancer tissue specimens collected by the surgical removal of stathmin protein expression in primary liver tissue by immunohistochemical staining using the monoclonal antibody was prepared. The immunohistochemical staining method reference the SABC kit manual steps to operate, the control group with PBS instead of primary antibody. 2.2 stathmin monoclonal anti-human antibody combined with paclitaxel growth inhibition of human hepatoma cell the 2.2.1 cells cultured human hepatoma cell line HepG2 cells 2 incubator at 37 ° C, 5% of the CO _{culture, culture medium RPMI-1640 containing 10% heat-inactivated fetal calf serum containing 1% of the double antibody every 3 ~ 4 d passaged 1 time, and take the logarithmic growth phase cells used for experiments. 2.2.2 inverted microscope HepG2 cell morphology changes take HepG2 cells in logarithmic growth phase, 2 × 10 ^{5 / seeded in 6-well plates, 4 h after adding different concentrations of stathmin monoclonal antibody different concentrations of paclitaxel, and the combination of medication, group processing 24,48,72,96 h continuous morphological changes were observed under an inverted microscope and shooting record cell morphology. 2.2.3 MTT assay stathmin monoclonal antibody combined with paclitaxel on HepG2 cell growth inhibition of HepG2 cells in logarithmic growth phase, 5 × 10 ^{3 / were seeded in 96-well plates, 37 ° C, 5% CO ₂ cultured for 4 h after the addition of drugs. Experimental groups: stathmin monoclonal antibody group, paclitaxel combination therapy group, with another the dosing control group, each dose set five wells. Added to each well were cultured 24,48,72,96 h MTT, cultured for 4 h. Termination of culture, the supernatant was removed, each well add 150μl DMSO, full melting of crystals OD value of each well was measured using a microplate reader, the calculation of cell proliferation inhibition rate. 2.2.4 The statistical analysis SPSS13.0 software processing system for statistical analysis. Different concentrations of anti-human stathmin monoclonal antibody, different concentrations of paclitaxel, and combination of medication group were compared HepG2 cells inhibition rate, the interaction effect using factorial analysis of variance analysis, the same concentration pairwise comparison between different time, at the same time under different concentrations between any two One-way ANOVA analysis. P <0.05 was considered statistically significant. Results of a successful screening and preparation of the two anti-stathmin monoclonal antibody cell lines. Second, by indirect ELISA two light chains are κ monoclonal antibody culture supernatant titer of 1:512 and 1:256, respectively, in accordance with the product specification HyCultBiotechnology obtained 2 cells secrete the monoclonal antibodies are IgG1 chain. Identified by Western-blot, anti-stathmin 2 monoclonal antibody cell culture supernatant of both the human hepatoma cell line (HepG2 and QGY7703), specifically react. Identified by immunohistochemistry, two anti-sathmin monoclonal antibody precipitate with primary hepatocellular carcinoma after reaction can produce brown or tan precipitation, mainly located in the cytoplasm of liver cancer cells, negative controls were brown or tan. Anti-stathmin monoclonal antibody, paclitaxel alone and associated with significantly inhibited HepG2 cells gradually increased with the increase of drug concentration and prolonged duration of action, and this inhibition, and showed a certain time dose dependencies; associated with a stronger inhibition than single-use display, indicating that there are obvious synergies between the two. Conclusion In this study, the use of hybridoma technology successfully screened and prepared two anti-stathmin monoclonal antibody cell lines, and its specific identification of two monoclonal antibodies are prepared by the Western-blot and immunohistochemical identification and human hepatoma cell lines and primary liver cancer pathological specimens react specifically with the preparation of monoclonal antibodies specific for of stathmin. Further anti-stathmin monoclonal antibody combined with paclitaxel, that stathmin monoclonal antibody anti-human, paclitaxel alone and in combination with the proliferation of HepG2 cells inhibition were in a time and concentration-dependent growth inhibition of human hepatoma cells was measured using the MTT assay With the increase of the concentration and prolonged duration of action, the inhibition of cell proliferation gradually enhanced, combined with stronger growth inhibition than single-use display, prompting interactive synergy between the two.}}}}

Preparation and Identification of Monoclonal Antibody Against Human Stathmin and Inhibitory Effect of the McAb on Hepatoma Carcinoma Cells

Abstract

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