嵌合抗原受体设计的NK细胞在多发性骨髓瘤中的应用研究

Application of Chimeric Antigen Receptor-Modified NK Cells in Multiple Myeloma

目的:通过建立CAR(CD138-CD28-CD3ζ)-NK细胞,探讨CAR(CD138-CD28-CD3ζ)-NK细胞对多发性骨髓瘤的抗肿瘤作用。方法:把抗CD138scFv-CD28-CD3连接到pc DNA3.1质粒,使用三质粒慢病毒包装系统转染293T细胞,收集病毒后感染NK92MI细胞系,应用嘌呤霉素筛选阳性细胞株;通过RT-PCR及Western blot检测CAR在NK92MI细胞系的表达,流式细胞术检测NK92MI细胞分泌细胞因子cd107a的能力。结果:经过基因工程技术,体外构建了CAR基因片段,基因测序验证基因序列正确。通过病毒包装技术,获得表达该片段的病毒株。经过病毒转染实验,获得表达CAR融合蛋白的稳转NK细胞株。PCR及Western blot证实,CAR融合蛋白在NK细胞表达。细胞杀伤实验证实CARNK细胞分泌细胞因子cd107a的能力明显优于对照组,具有明显的杀伤多发性骨髓瘤细胞的效应。结论:CAR能在体外构建并在NK92细胞表面表达,CARNK细胞能杀伤表达CD138抗原的多发性骨髓瘤细胞株,从而发挥抗骨髓瘤效应。

Objective： To explore the killing effect of CAR（ CD138-CD28-CD3ζ）-NK cells on myeloma cells through construction of CAR（ CD138-CD28-CD3）-NK cells.Methods： The anti CD138scFv-CD28-CD3 zeta plasmid pcDNA3.1 was constructed,which then together with 3 plasmid lentiviral packaging system were transfected into 293 T cells,the virus was collected.Furthermore,in order to get the stably transfected cell line,the NK92MI cell line was infected by the virus,then the positive cells were screened by puromycin.The expression of the CARNK cells were verified by RT-PCR and Western blot.At last the ability of secreting cytokine CD107 a was detected by flow cytometry,and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells.Results： Gene fragment of the CAR（ anti CD138scFv-CD28-CD3ζ） was constructed successfully by gene engineering technique in vitro,and the gene sequence was verified to be correct by sequencing.By virus packaging technology,the virus expressing the protein of the CAR was obtained.PCR and Western blot verified the expression of CAR fusion protein on the sufurce of NK cells.The cell killing experiment confirmed that the CAR-NK cells possessed the ability to secrete cytokine CD107 a superior to control cells and showed the obvious killing effect on multiple myeloma cells.Conclusion： The CAR can be constructed in vitro,and express on NK92 cells.The CAR-NK cells can kill the multiple myeloma cells expressing CD138 antigen,thereby plays an antimyeloma effect.