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-  2018 

CART免疫疗法中慢病毒拷贝数检测的实验研究 Detection of Lentivirus Copy Number in the Chimeric Antigen Receptor T Cell Immumotherapy

Keywords: CART免疫治疗,慢病毒拷贝数,TaqMan实时定量技术

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Abstract:

目的:利用特异探针检测慢病毒拷贝数,在CART免疫治疗中准确区分携带不同靶点的CART细胞在体内的增殖情况。方法:采用分子克隆技术构建含CD19和CD22CAR结构的慢病毒重组质粒;通过流式和ELISA技术检测两种CART细胞在体外与Raji共培养时的杀瘤效率;通过TaqMan技术检测293T细胞及病人基因组中的病毒拷贝数。结果:CD19和CD22的慢病毒载体成功构建,并收集具有高滴度的病毒颗粒;体外研究结果显示感染病毒的T细胞(CD19-CART和CD22-CART)分别与Raji共培养组的杀瘤效率和IL-6释放水平均显著(P<0.05)或极显著(P<0.01)高于对照组,在293T细胞中,转染了病毒与重组质粒组的慢病毒拷贝数均显著高于未转染组(P<0.05);CD19-CART和CD22-CART序贯回输20min后,均可在体内检测到102-104个病毒拷贝,到第10天显著上升(P<0.05),随后变化平稳,且均高于初始回输水平。结论:跟踪监测病毒拷贝数,能确定特定CART细胞在体内的存续时间,为选择合适的治疗时机提供实验依据

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