CAJ | 학술논문

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/mL。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。
목적：연구자삼순、순박대인식관암EC9706세포NKG2D배체표체급CIK세포살상활성적영향，탐토상관분자궤제。방법：MTT법측정자삼순、순박대EC9706세포적24 h반수억제농도（IC50）。류식세포의검측1/2 IC50농도자삼순、순박작용전、후EC9706세포NKG2D배체적표체。유산탈경매석방법검측효파비20：1、30：1시，CIK세포대1/2 IC50농도자삼순、순박작용전、후EC9706세포적살상활성。형광정량PCR법검측1/2 IC50농도자삼순、순박작용EC9706세포24 h전、후DNA손상수복기인（ATM、ATR、CHK1、CHK2、P53）표체적변화。결과：자삼순、순박적24 h반수억제농도분별위10、5μg/mL。1/2 IC50농도자삼순작용24 h후，EC9706세포MICB、ULBP2、ULBP3표체균명현증강（P<0.05），MICA、ULBP1표체무현저성변화（P>0.05）；1/2 IC50농도순박작용24 h후，EC9706세포MICA、MICB、ULBP2、ULBP3표체균명현증강（P<0.05），ULBP1표체무현저성변화（P>0.05）。효파비20：1、30：1시，CIK세포대1/2 IC50농도자삼순、순박작용후적EC9706세포적살상활성균명현증강（P<0.05）。1/2 IC50농도자삼순작용24 h후，DNA손상수복기인표체균무현저성변화（P>0.05）；1/2 IC50농도순박작용24 h후，ATM、ATR、CHK1、CHK2기인표체균명현증가（P<0.05），P53기인표체무현저성변화（P>0.05）。결론：순박、자삼순균가증강CIK세포적살상활성，기분자궤제가능여격활DNA손상수복기인，진이증가NKG2D배체표체유관。
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.