目的：探讨舒尼替尼促进肝癌细胞自然杀伤细胞2族成员D配体（natural killer group 2 member D ligands，NKG2DLs）表达及提高NK细胞杀伤肿瘤细胞的作用。方法：常规体外培养HepG2细胞，免疫磁珠法从健康志愿者外周静脉血中分选NK细胞，流式细胞技术检测NK细胞纯度及1 μmol/L舒尼替尼孵育24 h前后肝癌细胞NKG2DLs表达率；LDH释放测定法检测NK细胞对药物处理前后靶细胞的杀伤活性，实时荧光定量PCR检测药物处理前后HepG2细胞NKG2DLs mRNA的表达情况。结果：分选后NK细胞(CD3-CD16+CD56+细胞)的纯度达78%以上；经舒尼替尼处理后靶细胞MHC-Ⅰ类链相关分子A或B（MHC class Ⅰ-related chain molecules A/B, MICA/MICB）、人巨细胞病毒糖蛋白UL16结合蛋白（UL16-binding proteins，ULBPs）的表达率均有升高，以MICA、MICB和ULBP2升高最明显（F=17.73，P=0.000）。当效靶比为10∶1、20∶1时，NK细胞对舒尼替尼处理前后HepG2细胞的杀伤活性分别从(9.47±1.11)%、(20.45±1.94)%上升到(28.88±123)%、(44.93±1.57)%，舒尼替尼处理前后NK细胞对靶细胞杀伤活性的差异有明显统计学意义(P<0.05)。舒尼替尼处理HepG2细胞后，MICA、MICB和ULBP2 mRNA表达水平明显升高（F=62.628，P=0.000）。结论：舒尼替尼能选择性诱导肿瘤细胞高表达NKG2DLs(MICA/B和ULBP2)，并增强NK细胞杀伤活性。

Objective:To investigate the effect of sunitinib on natural killer group 2 member D ligands (NKG2DLs) expression and NK-mediated cytotoxicity in human hepatocellular carcinoma cells. Methods: HepG2 cells were cultivated by routine method. Human NK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of the isolated NK cells and the expression levels of NKG2DLs on HepG2 cells. The cytotoxic effect of NK cells against HepG2 was assessed with LDH releasing assay. The expressions of NKG2DL mRNAs in HepG2 cells was quantitated by RT-qPCR. Results: More than 78% of the isolated cells were CD3-CD16+CD56+, indicative of NK cells. After sunitinib treatment, the expressions of multiple NKG2DLs on HepG2 cells were increased, especially that of MICA, MICB and ULBP2. At the E∶T ratio of 10∶1 and 20∶1, the cytotoxic effects of NK cells against HepG2 cells were increased from (9.47±1.11)% and (20.45±1.94)% in the untreated groups to (28.88±1.23)% and (44.93±157)% in the sunitinib treatment groups (P<0.05). The expression of MICA, MICB and ULBP2 mRNA in HepG2 cells was also significantly elevated after treated with sunitinib. Conclusion: Sunitinib regulates the expressions of NKG2DLs (MICA/B and ULBP2) on HepG2 cells, which activates NK cells and is responsible for their enhanced cytotoxic action.

国家自然科学青年基金资助项目（No. 81302372，81300431）；南方医科大学珠江医院优秀中青年人才项目资助（No. 201207008)