Preparation Of BCL2/IGH-CAR T Cells And Its Biological Effects In Vitro

Objective:To preparate of BCL2/IGH-CAR~+T cells and explore the biological effects in vitro for the treatment of lymphoma to seek new ideas and methods.Methods:1?Using pHBLV-BCL2/IGH ScFv-CD28-CD3?,psPAX2,pMD2.G three plasmids co-transfected 293T cells to prepare the lentivirus vector carrying the gene of BCL2/IGH ScFv-CD28-CD3?.2?Extraction of healthy adult male donor peripheral blood;Isolate peripheral blood mononuclear cells with lymphocyte separation solution;The total T cells of peripheral blood with CD3~+were selected by magnetic bead separation technology.The activation of CD3~+T cells was activated by activating magnetic beads with anti-biotin markers.3?BCL2/IGH-CAR~+T were prepared by using lentivirus with a target gene to infect CD3~+T cells.Fluorescence microscopy and flow cytometry were used to detect the positive rate of green fluorescent protein expression in cells.The expression of CAR molecules in CAR~+T cells was analyzed by Western blot.4?The expression of BCL2 protein in two lymphoma target cells was identified by RT-PCR and WB.5?BCL2/IGH-CAR~+T cells were cultured with DOHH2 and jeko-1 cells respectively to detect the biological effects of CAR~+T in vitro.The differences in the secretion of INF-?and IL-2 cytokines were detected by ELISA kit.The difference of the killing activity of CAR~+T on two kinds of target cells was detected by the LDH detection kit.Results:1?The lentiviral expression vector carrying BCL2/IGH-CAR gene was successfully packaged;2?CD3~+T cells were successfully infected with lentiviral vectors prepared by early experiments,and CAR~+T cells with a positive rate of 62.7%were prepared;3?Both DOHH2 and jeko-1 lymphoma cell lines have the expression of BCL2 protein,and the BCL2 of DOHH2 cells is overexpressed.The BCL2 protein in jeko-1 cell line was low expression;4?The CAR~+T cells prepared in this experiment have cytotoxic activity on DOHH2 overexpressing BCL2 protein and low expression of jeko-1 lymphoma cell line;their ability of stimulating cytokine secretion and killing activity and the expression level of BCL2 protein in target cells Related;CAR~+T cells on the two target cells in vitro biological effects were statistically different.Conclusion:The experiment of BCL2/IGH-CAR~+T in vitro biological effects of preliminary research shows that the target BCL2 as the tumor cell surface antigen,preparation of BCL2/IGH-CAR~+T cells can effectively targeted BCL2 protein,and effective activation of BCL2/IGH-CAR~+T cells,stimulate the secretion of cytokines INF-?and IL-2.To improve the selective killing activity of T cells on tumor cells.