目的： 研究经IL-7和IL-21修饰后的NK-92MI细胞的增殖和细胞毒性及其对正常人外周血中T细胞的影响。 方法： 通过基因工程将IL-7和IL-21基因片段构建到电转载体上，通过电转的方式构建了NK-92MI/IL-21和NK-92MI/IL-7&21细胞。 以细胞计数法和CFSE/7-AAD流式术分别对NK-92MI、NK-92MI/IL-21和NK-92MI/IL-7&21细胞体外增殖活性和细胞毒性进行 比较。将正常人PBMC与NK-92MI、NK-92MI/IL-21和NK-92MI/IL-7&21细胞体外共培养后，用流式细胞术检测PBMC中T细 胞的表型变化；同时检测正常人活化后的T细胞与NK-92MI、NK-92MI/IL-21和NK-92MI/IL-7&21细胞共存时相互的细胞毒性以 及对肿瘤细胞的毒性比较。 结果： 成功构建了高表达IL-21的NK-92MI/IL-21细胞和同时高表达IL-7和IL-21的NK-92MI/IL-7& 21细胞；NK-92MI、NK-92MI/IL-21和NK-92MI/IL-7&21细胞对Jurkat和K562细胞的细胞毒性没有变化，但是NK-92MI/IL-21和 NK-92MI/IL-7&21细胞相对于亲本细胞NK-92MI的体外增殖活性有所提高；NK-92MI/IL-21和NK-92MI/IL-7&21细胞对PBMC 中T细胞的活化有一定的促进作用；活化后的T细胞与NK-92MI、NK-92MI/IL-21和NK-92MI/IL-7&21细胞相互之间几乎无细胞 毒性；同时，当T细胞和3种NK细胞共存时，T细胞不影响NK细胞对K562细胞的细胞毒性。 结论： 基因工程修饰的NK-92MI/ IL-21和NK-92MI/IL-7&21细胞体外增殖活性有所提高，它们对外周血T细胞的活化有一定的刺激和促进作用；T细胞和NK- 92MI细胞之间无细胞毒性，同时活化T细胞的存在不影响NK-92MI细胞的细胞毒性。

To investigate whether the proliferation and cytotoxicity of NK-92MI cells can be improved by IL-7 and IL-21 genes modification, and determine the effects of this genetically modified NK-92MI cells on T cells from normal human peripheral blood. Methods：IL-7 and IL-21 gene fragments were constructed into electroporation vector by genetic engineering method, and NK- 92MI/IL-21 and NK-92MI/IL-7&21 cells were constructed by electroporation transfection. The in vitro proliferation and cytotoxicity of NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells were measured by cell count and flow cytometry assays. Then, normal human PBMCs were co-cultured with NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells in vitro respectively, and the phenotype change of T cells was measured by flow cytometry. In addition, the cytotoxicity between the activated T cells and three NK-92MI cell lines (NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells) as well as the cytotoxicity of the three NK-92MI cells on tumor cells after co-incubation with activated T cells were detected. Results: NK-92MI/IL-21 cell line (highly expressing IL-21) and NK-92MI/IL-7& 21 cell line (highly expressing both IL-7 and IL-21) were successfully constructed. The toxicity of NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells on Jurkat and K562 cells showed no difference, while the proliferation of NK-92MI/IL-21 and NK-92MI/IL-7&21 cells was increased compared with NK-92MI cells. Furthermore, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells promoted the activation of T cells to a certain degree, and the activated T cells showed merely no cytotoxicity on NK-92MI, NK-92MI/IL-21 and NK-92MI/IL- 7&21 cells; Meanwhile, the activated T cells did not affect the cytotoxicity of the three NK cells (NK-92MI, NK-92MI/IL-21, and NK- 92MI/IL-7&21 cells) on K562 cells under their co-existence. Conclusion: The in vitro proliferation of NK-92MI/IL-21 and NK-92MI/ IL-7&21 cells were enhanced after gene modification, which could also stimulate and promote the activation of T cells from peripheral blood. The cytotoxicity assay showed that the activated T cells had no cytotoxicity on NK-92MI, NK-92MI/IL-21, and NK-92MI/IL-7& 21 cells. Meanwhile, the presence of the activated T cells did not affect the cytotoxicity of NK-92MI cells.

国家重点研发计划(No.2016YFC1303403)；国家自然科学基金资助项目(No.31471283)；江苏高校血液学协同创新中心(No. XYXT2015304)；江苏高校优势学科建设工程资助项目