顺铂和紫杉醇对CIK细胞杀伤食管癌细胞活性的影响及其分子机制研究

Effect of cisplatin and paclitaxel on the cytotoxicity of cytokine-induced killer cells on esophagus carcinoma and its molecular mechanisms

目的:研究紫杉醇、顺铂对人食管癌EC9706细胞NKG2D配体表达及CIK细胞杀伤活性的影响,探讨相关分子机制。方法:MTT法测定紫杉醇、顺铂对EC9706细胞的24 h半数抑制浓度(IC50)。流式细胞仪检测1/2 IC50浓度紫杉醇、顺铂作用前、后EC9706细胞NKG2D配体的表达。乳酸脱氢酶释放法检测效靶比20:1、30:1时,CIK细胞对1/2 IC50浓度紫杉醇、顺铂作用前、后EC9706细胞的杀伤活性。荧光定量PCR法检测1/2 IC50浓度紫杉醇、顺铂作用EC9706细胞24 h前、后DNA损伤修复基因(ATM、ATR、CHK1、CHK2、P53)表达的变化。结果:紫杉醇、顺铂的24 h半数抑制浓度分别为10、5μg/m L。1/2 IC50浓度紫杉醇作用24 h后,EC9706细胞MICB、ULBP2、ULBP3表达均明显增强(P<0.05),MICA、ULBP1表达无显著性变化(P>0.05);1/2 IC50浓度顺铂作用24 h后,EC9706细胞MICA、MICB、ULBP2、ULBP3表达均明显增强(P<0.05),ULBP1表达无显著性变化(P>0.05)。效靶比20:1、30:1时,CIK细胞对1/2 IC50浓度紫杉醇、顺铂作用后的EC9706细胞的杀伤活性均明显增强(P<0.05)。1/2 IC50浓度紫杉醇作用24 h后,DNA损伤修复基因表达均无显著性变化(P>0.05);1/2 IC50浓度顺铂作用24 h后,ATM、ATR、CHK1、CHK2基因表达均明显增加(P<0.05),P53基因表达无显著性变化(P>0.05)。结论:顺铂、紫杉醇均可增强CIK细胞的杀伤活性,其分子机制可能与激活DNA损伤修复基因,进而增加NKG2D配体表达有关。

Objective：To explore the effect of paclitaxel （PTX） and cisplatin （DDP） on the expression of NKG2D ligands of hu-man esophagus carcinoma cell EC9706 and on the cytotoxicity of cytokine-induced killer （CIK） cells, as well as to discuss its molecu-lar mechanisms. Methods： The half maximal inhibitory concentration （IC50） values of PTX and DDP against EC9706 cells for 24 h were measured by MTT assay. The expression levels of NKG2D ligands （MICA, MICB, ULBP1, ULBP2, and ULBP3） on the EC9706 cell surface before and after 24 h culture with 1/2 IC50 of PTX or DDP were assayed by flow cytometry. Cytotoxicity of CIK cells against EC9706 cells before and after 24 h culture with 1/2 IC50 PTX or DDP was analyzed by lactate dehydrogenase release assay at an effector to target cell ratio （E：T） of 20：1 and 30：1, respectively. The expression levels of DNA damage repair genes （ATM, ATR, CHK1, CHK2, and p53） of EC9706 cells before and after 24 h incubation with 1/2 IC50 PTX or DDP were detected by quantitative fluorescent PCR. Results：The IC50 values of PTX and DDP were 10 and 5μg/mL, respectively. MICB, ULBP2, and ULBP3 on EC9706 cells were upregulated after 24 h culture with 1/2 IC50 PTX （P〈0.05）, and the expression levels of MICA, MICB, ULBP2, and ULBP3 were higher after 24 h culture with 1/2 IC50 DDP （P〈0.05）. Cytotoxicity of CIK cells against EC9706 cells cultured with 1/2 IC50 of PTX or DDP at E：T of 20：1 and 30：1 was significantly enhanced compared with those untreated （P〈0.05）. The expression levels of DNA damage repair genes did not significantly increase after 24 h treatment with 1/2 IC50 PTX （P〉0.05）, whereas ATM, ATR, CHK1, and CHK2 were over-expressed after 24 h treatment with 1/2 IC50 DDP （P〈0.05）. Conclusion：PTX or DDP can enhance the susceptibility of EC9706 cells to CIK cell-mediated lysis by upregulating the expression of NKG2D ligands through activating DNA damage repair genes.