| [Objective] The expression of CD20 antigen in B lymphoma cells is a recognized therapeutic target. But CD20 antibody serum half-life is short and easy to drug resistance, can not be completely kill tumor cells. Through the immune escape mechanism, lymphoma makes T cells lose the immune monitoring function. Recent experiments confirmed that CD137, CD28 can activate the T cells in vitro, so that it can recover the proliferation ability and anti tumor ability in vivo. In this study, the chimeric antigen receptor which is composed of CD20 antibody and CD137, CD28 antibody was transfected and activated by T cells, which made it have the function of targeting killing and immune monitoring to lymphoma cells.[Methods] I used gene synthesis technology to synthetise CD20-28-137-TCR-ζ gene sequence and send it to the gene sequencing company to detect, and compared to the gene sequence map for detecting the correctness of gene synthesis. Constructing the slow virus vector, the gene sequence was transfected into the slow virus vector. In order to get efficient and stable slow virus vector, I detected slow virus titer. I used COBE cell apheresis machine to collect peripheral blood cells of lymphoma patients, and used lymphocyte separation liquid to separate the cells for removaling of the B cells, lymphocytes. I cultured the lymphocytes as CIK cells, CD3, IFN-, IL-2, stimulate T cell growth, in order to obtain more pure to T cells. CD3, IFN-, IL-2 cytokines will be added, in order to stimulate the growth of T cells, and to get a more pure T cells. T cells were transfected with a highly efficient and stable slow virus vector which was previously constructed, to synthesize CAR-T-20 cells, and then to test the test. LDH method was used to determine the cytotoxicity of CAR-T cells. CAR cells and lymphoma cells were mixed together in accordance with a certain proportion for detecting the cytotoxic effect of CAR-T cells on lymphoma cells. I would determine the proportion of CAR-T and lymphocytes with the maximum killing effect. To construct a mouse model of lymphoma, the CAR-T cells were injected into the mice. I would observe the changes of tumor size in mice, and make a record to judge the effect of CAR-T on lymphoma in vivo.[Results] Through the detection of gene sequences, CD20-28-137-TCR-ζ gene sequence consistent with the objectives and they have the same length. It shows that the gene was successfully constructed. The agarose gel electrophoresis was used to detect the lentiviral vector. The clear bands and the same position indicated that CD20-28-137-TCR-ζ gene was successfully loaded in lentiviral vectors After that, the virus vector was packaged and the titer was measured. The lentiviral vector was successfully packaged by observing the GFP. The titer of the virus was calculated to be 8.25* 108TU/ML. T cells were transfected with the lentiviral vector. According to the number of T cells before and after transfection, the transfection efficiency was 29%. LDH tests were carried out in a total of four proportions, and the results showed that the ratio of CAR-T cells to lymphoma cells in the case of 10:1, the most lethal effect on lymphoma. Mouse model was constructed in accordance with 10 rats in each group, and divided into three groups which is the blank control group, negative control group and positive control group. The experimental results show that the positive control group of tumor growth has obvious inhibitory effect. Negative control group and blank control group, the tumor growth rate is almost the same.[conclusion] The experiments in vitro fully confirmed that the CAR-T cells have a killing effect in lymphoma of patients, and when the ratio of CAR-T cells to lymphoma cells reached 10:1, the killing effect was the best. The experiments in vivo proved that CAR-T cells had a certain inhibitory effect on the growth of lymphoma. In summary, it is preliminarily proved that CAR-T cells have a certain therapeutic effect on the treatment of B cell lymphoma. |
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