顺铂和氟尿嘧啶增强食管癌细胞NKG2D配体的表达及CIK细胞的杀伤活性

Cisplatin and 5-fluorouracil Enhance Expression of NKG2D Ligands and CIK Cells Killing Effect of Human Esophagus Carcinoma Cell EC9706

目的研究顺铂(Cisplatin,DDP)和氟尿嘧啶(5-fluorouracil,5-Fu)对人食管癌EC9706细胞NKG2D配体表达及CIK细胞杀伤活性的影响。方法 MTT法测定DDP、5-Fu的50%抑制浓度(IC50);以1/2 IC50浓度DDP、5-Fu作用EC9706细胞72 h,RT-PCR检测DNA损伤修复系统相关信号分子的表达。流式细胞仪检测DDP、5-Fu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时,CIK细胞对DDP、5-Fu作用前、后EC9706细胞的杀伤活性。结果 DDP、5-Fu的IC50分别为5μg/ml、10μg/ml。DDP、5-Fu可上调DNA损伤修复系统相关信号分子mRNA的表达。DDP与EC9706细胞共孵育72 h后,EC9706细胞MICA、MICB、ULBP2、ULBP3表达较DDP作用前明显增强(P<0.05),ULBP1无明显变化(P>0.05)。5-Fu与EC9706细胞共孵育72 h后,EC9706细胞MICA、ULBP2、ULBP3表达明显增强(P<0.05),MICB、ULBP1无明显变化(P>0.05)。效靶比20∶1时,CIK细胞对EC9706细胞的杀伤活性为(37.08±0.62)%,CIK细胞对1/2 IC50DDP、1/2 IC505-Fu作用后的EC9706细胞杀伤活性分别为(59.33±2.10)%、(52.44±0.97)%,与作用前相比差异均有统计学意义(P<0.05)。结论 DDP、5-Fu通过激活DNA损伤修复系统相关信号分子,提高EC9706细胞NKG2D配体的表达,从而增强EC9706细胞对CIK细胞杀伤的敏感度。

Objective To explore the effects of Cisplatin（DDP） and 5-fluorouracil（5-Fu） on the expression of NKG2D ligands of human esophagus carcinoma cell EC9706 and cytokine-induced killer（CIK） cells cytotoxicity.Methods The IC50 of DDP,5-Fu against EC9706 cells were measured by MTT assay.Expressions of signal pathway molecules involved in DNA damage and repair system were detected by RT-PCR.The expression of NKG2D ligands（MICA,MICB,ULBP1,ULBP2,ULBP3） were analyzed by flow cytometery.Cytotoxicities of CIK cells against EC9706 cells before and after cultured by 1/2 IC50 DDP or 5-Fu were analyzed by LDH releasing assay at effector-to-target cell ratio（E∶T） of 20∶1.Results DDP,5-Fu could decrease the proliferation and survival rate of EC9706 cells,the IC50 was 5 μg/ml and 10 μg/ml,respectively,which increased mRNA expressions of signal pathway molecules involved in DNA damage and repair system.MICA,MICB,ULBP2,ULBP3 on EC9706 cells were over expressed after 72 h cultured with 1/2 IC50 DDP,while expression of MICA,ULBP2,ULBP3 were higher after treated by 1/2 IC50 5-Fu.Cytotoxicity of CIK cells against EC9706 cells cultured by 1/2 IC50 DDP,1/2 IC50 5-Fu were（37.08±0.62）%,（59.33±2.10）%,（52.44±0.97）%,respectively.Cytotoxicity of CIK cells against EC9706 cells cultured by either 1/2 IC50 DDP or 1/2 IC50 5-Fu was significantly enhanced.ConclusionThe results indicate that DDP or 5-Fu can enhance the susceptibility of EC9706 cells to CIK cells-mediated killing effect by upregulating the expressions of NKG2D ligands through activating signal pathway molecules involved in DNA damage and repair system.