The Establishment Of HGPRT-Deficient Cell Lines And Its Fusion With Human Lymphocyte Cells

The article aimed at the establishment of HepG2 and Hela cell lines with hypoxanthine guanine phosphoribosyl transferase(HGPRT) deficiency, using the chemical mutagen of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) to induce the cells,and conduct the cell fusion with the cells above and human lymphocyte.And we got two kinds of cells with HGPRT deficiency,also got two kinds of fusion cells.The separation of human lymphocyte was done with the tonsil of surgical ablation and we got a mixture of human lymphocytes.The separation of T,B lymphocytes was proceeded with nylon column.then the identification is carried out with Erythrocyte rosette test and Enzyme-Linked Immunosorbnent Assay (ELISA). The formation rate of erythrocyte rosette test is 59.4%,and the denisity of CD23 of cell surface antigen is 30 pg/ml-60 pg/ml.The mixed cells were observed with microscope,with regular round shape,brightness,the phenomenon of aggregation growth after culturing 4-5days.The shape of T,B lymphocytes has no significant differences in appearance.Obsereved in high magnification, the size of T lymphocytes is a bit smaller than those of B lymphocytes. B lymphocytes cultured in vitro,the decline phase of B lymphocytes is in the seventh day, then the cells quickly dies,and all cells die in the ninth day. T lymphocytes can be cultured in vitro for 20 days, also with decline phase and gradual death.The HepG2 cells were cultured with N-methyl- N-nitro-N-nitrosoguanidine (MNNG) for mutation and selected by increasing concent ration of 6-thioguanine (6-TG). The 6-TG resistant mutant cells were cultured in the medium containing 6-TG or HAT.After four months of screening, a deficient cell line was established. It could grow vigorously in the medium containing 20μg/ml 6-TG, while it all died within 12 days in the medium containing HAT. Karyotypic analysis showed that the number of the chromosomes of the deficient cells are between 32-88, with the modal number being 48.The 6-TG resistant and aminopterin-sensitive character of the mutant cell line reveals that it is deficient in HGPRT. The cell line established in this study will be a good parent cell line for preparation of hybridoma in the future.The cell fusion was done with fresh human lymphocytes and HepG2 and Hela cells with HGPRT deficiency,in the the logarithmic phase,with PEG fusion agent.We selected the hybridoma in the selective medium of HAT, cloning the cells with limiting dilution analysis(LDA),then we got the hybridoma accordingly.After the selection in the selective medium,the cytoplasm of the hybridoma expands, with multicore phenomenon, irregular cell shape,clear and increased granule.When the hybridoma begins cell division,their form returns to normal.The establishment of the hybridoma makes the human lymphocytes cultured in vitro unlimited,and make some basic for the research of the production of the human monoclonal antibody in vitro.