[摘要] 目的：评价体外扩增的人NK细胞能否作为呼肠孤病毒的运载细胞并探讨其临床转化应用价值。方法：体外扩增人NK细胞，用NK细胞装载呼肠孤病毒，激光共聚焦显微镜观察呼肠孤病毒与NK细胞结合的部位。NK细胞运载呼肠孤病毒（Reo-NK）到达肿瘤细胞后，CCK-8 法检测在中和抗体的存在下病毒发挥的溶瘤作用；qPCR法检测肿瘤细胞内病毒RNA的相对表达量。CCK-8 法检测Reo-NK对结直肠癌KRAS基因突变型（DLD-1）和野生型（CaCo-2、HT29）细胞株的杀伤效应，ELISA 双抗体夹心法检测NK细胞释放的穿孔素水平。结果：呼肠孤病毒主要结合于NK细胞膜上，在人AB型血清的存在下体外扩增的NK细胞可将呼肠孤病毒传递到肿瘤细胞，且传递后的呼肠孤病毒仍具有显著溶瘤活性（P<0.01）；同时检测到肿瘤细胞内病毒RNA的表达量显著增高（P<0.01）。与NK组相比，Reo-NK组对KRAS基因突变型和野生型结直肠癌细胞株的细胞毒作用均显著增强（P<0.05），穿孔素的释放均明显增加（P<0.05）。结论：体外扩增的人NK细胞适合作为呼肠孤病毒的运载细胞，且呼肠孤病毒能够增强NK细胞的细胞毒作用，两者联合后对结直肠癌细胞可发挥更强的杀伤作用，因而具有重要的临床转化应用价值。

[Abstract] Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3-CD56+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus (Reo-NK)to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant (DLD-1) and KRAS wild type (CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity (P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly in-creased over time (P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild (all P<0.05), and significantly increased the release of perforin (all P<0.05). Conclusion:In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells，that has important clinical application value.

国家自然科学基金资助项目（No. 81860542）；中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助（No.2017PT31042）; 贵州省普通高等学校科技拔尖人才支持计划（黔教合KY字[2018]049）；贵州省应用基础研究计划重大专项（黔科合J 重大字[2015]2003）；贵州省科技厅社会发展攻关项目（黔科合SY字[2013]3010 号）