靶向Robo1嵌合抗原受体修饰的NK92细胞对神经胶质瘤及神经母细胞瘤细胞的杀伤效果

Killing effect of Robol targeted Chimeric Antigen Receptor modified NK92 cells against glioma and neuroblastoma cells

目的研究Roundabout1（Robo1）-CAR-NK92细胞在体外对神经胶质瘤细胞U87-MG及神经母细胞瘤细胞SH-SY5Y的体外杀伤以及探讨白细胞介素-15（IL-15）、IL-21和地塞米松对Robo1-CAR-NK92细胞增殖、活性和杀伤的影响以优化培养方案。方法用慢病毒转染的方法构建Robo1-CAR-NK92细胞后分选扩增，并用流式细胞术检测阳性率。用流式细胞术的方法检测U87-MG及SH-SY5Y细胞表面Robo1的表达。用CCK-8试剂盒和动态活细胞成像的方法检测Robo1-CAR-NK92及NK92细胞对靶细胞的体外杀伤。用流式多重微珠阵列法检测杀伤过程中分泌的细胞因子含量。用锥虫蓝染色的方法检测经25 ng/ml的IL-15，25 ng/ml的IL-21和（或）50 nmol/L的地塞米松处理3 d后的Robo1-CAR-NK92及NK92细胞的计数及活性率。结果构建Robo1-CAR-NK92细胞并经分选扩增后检测阳性率为98.89%。流式细胞术检测结果显示，Robo1在U87-MG及SH-SY5Y细胞上表达的阳性率分别为88.14%，99.75%。Robo1-CAR-NK92细胞对靶细胞的杀伤率明显高于NK92的杀伤率（P〈0.05）。Robo1-CAR-NK92细胞在杀伤U87-MG细胞过程中分泌的IL-6、IL-10、TNF-α和IFN- γ较NK92细胞明显增多（P〈0.05）。IL-15能促进Robo1-CAR-NK92的增殖和活性而IL-21则起相反作用。IL-21、IL-15＋IL-21能增强NK92和Robo1-CAR-NK92细胞的杀伤效果。IL联用地塞米松能增强NK92和Robo1-CAR-NK92细胞的增殖和活性，但抑制二者的杀伤效果（除IL-15和地塞米松联用不影响Robo1-CAR-NK92细胞的杀伤效果外）。结论与NK92细胞比，Robo1-CAR-NK92细胞对神经胶质瘤及神经母细胞瘤细胞有更强大的杀伤效果。在本试验时间内，IL-15联用地塞米松处理Robo1-CAR-NK92细胞是最有利于Robo1-CAR-NK92细胞体外增殖的培养方案。

ObjectiveTo study the cytotoxicity of Robo1-CAR-NK92 cells against U87-MG and SH-SY5Y cells, to explore the effects of IL-15, IL-21 and dexamethasone on the proliferation, survival and cytotoxicity of Robo1-CAR-NK92 cells and to optimize the culture protocol.MethodsRobo1-CAR-NK92 cells were constructed by lentivirus transfection.The Robo1 car positive cells were sorted, expanded and detected by flow cytometry.The levels of Robo1 expression in SH-SY5Y and U87-MG cells were examined by flow cytometry.The cytotoxicity of Robo1-CAR-NK92 or NK92 cells against target cells was tested by CCK-8 and live cell imaging. The levels of cytokines in the supernatant of cultured cells during the cytotoxicity assay were quantified by the multiplex bead-array assay.NK92 and Robo1-CAR-NK92 cells （4×104/ml） were treated with 25 ng/ml of IL-15, 25 ng/ml of IL-21 and/or 50 nmol/L dexamethasone for 3 days and were stained with trypan blue to acquire the viable cell numbers and survival rates.ResultsRobo1-CAR-NK92 cells were constructed and tested 98.89% positive after sorting and expansion. While 88.14% of U87-MG cells were Robo1 positive, there were 99.75% of Robo1 positive SH-SY5Y cells.The specific lysis of Robo1-CAR-NK92 cells against target cells was significantly higher than that of NK92 cells （P〈0.05）. Robo1-CAR-NK92 cells obviously secreted more cytokines including IL-6, IL-10, TNF-α and IFN-γ than parental NK92 cells during cytotoxic activity against U87-MG cells （P〈0.05）. IL-15 significantly increased the proliferation and survival of Robo1-CAR-NK92 cells, but IL-21 played the opposite role.Remarkably, IL-21 and IL-15＋ IL-21 enhanced the cytotoxicity of NK92 and Robo1-CAR-NK92 cells.The combination of dexamethasone and interleukins dramatically promoted the proliferation and survival but obviously impaired the cytotoxicity of NK92 and Robo1-CAR-NK92 cells （except that IL＋ 15 and dexamethasone have no effect on the cytotoxicity of Robo1-CAR-NK92 cells）.ConclusionsCompared to parental NK92 cells, Robo1-CAR-NK92 cells exhibited more potent targeted killing against glioma and neuroblastoma cells.Collectively, treatment of IL-15 and dexamethasone was the optimized protocol for culture of Robo1 CAR NK cells during our experimental time.