抗CD19 CAR-T对K562^(CD19+)肿瘤细胞特异性杀伤作用的研究

The specific killing effect of anti-CD19 CAR-T cells on K562^(CD19+) tumor cells

目的构建表达抗CD19分子的嵌合抗原受体,制备抗CD19 CAR-T细胞,研究其对CD19表型细胞的特异性识别和杀伤效果。方法通过分子克隆方法,构建能够自剪切EGFP荧光报告的抗CD19 CAR分子,将其重组到慢病毒载体中并包装获得慢病毒。通过慢病毒递送的方式,将抗CD19 CAR过表达于primary-T细胞,流式细胞术分析确认表达后,通过ELISA检测CAR-T细胞IL-2分泌情况,最后用LDH释放法评价靶向杀伤作用。结果成功获得抗CD19 CAR的慢病毒递送载体;流式细胞术表明CAR分子在T细胞表面高效稳定表达;ELISA结果显示CAR-T细胞在靶细胞刺激下IL-2分泌水平显著升高(P<0.01);LDH结果显示CAR-T细胞特异性杀伤CD19表型细胞,且杀伤效果在E:T=10:1时达到50%以上,显著高于对照组(P<0.05)。结论成功获取了具有靶向CD19表型细胞的CAR-T细胞,能高效特异性杀伤肿瘤细胞。

Objective To construct a lentivirus vector expressing anti-CD19 chimeric antigen receptors and its engineered T cells and investigate the specific cytotoxicity of CAR-T cells on CD19 positive B-cell lymphoma cells.Methods Technology of molecular cloning was employed to construct anti-CD19 CAR with self-cleavage enhanced green fluorescent protein,and then to package lentivirus with packaging and envelope plasmids.Using lentivirus delivery system,anti-CD19 CAR were overexpressed on primary-T cells.Followed by the identify of anti-CD19 CAR expression on T cells using flow cytometry,ELISA and LDH assay were used to detect the IL-2 secretion and killing effect of the CAR-T cells,respectively.Results We successfully constructed the CD19-specific CAR gene recombined lentivirus and established CD19-specific CAR-engineered T cells.Results from flow cytometry evaluated with EGFP suggested that anti-CD19 CAR were stably and efficiently expressed on T cell surface.The secretion of IL-2 from CAR-T cells was significantly more than the primary-T cells(P<0.01),and the cell killing activity of CAR-T cells increased with the E:T change,especially the killing effect rate over 50%at E:T=10:1(P<0.05).Conclusion We successfully establish the CD19-specific CAR-engineered T cells with specifically and efficiently killing effect on the target cells.