三重靶向介导的Smac过表达对乳腺癌MDA-MB-231细胞凋亡和周期进程的影响

目的：利用基因重组技术获得三重靶向性Smac过表达的条件复制型腺病毒，探讨其对乳腺癌MDA-MB-231细胞凋亡和周期的影响。方法：利用重组技术构建Smac过表达载体pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K，与骨架载体pAdEasy在BJ5183（AdEasy-1+）菌中进行重组，获得Smac过表达的条件复制型腺病毒CRAd.pE-Smac，感染MDA-MB-231细胞后，利用化学试剂氯化钴模拟肿瘤细胞乏氧状态，设立对照组、CARd.pE-Smac组、乏氧组和CARd.pE-Smac+乏氧组，并根据是否进行4 Gy照射，每组分为未照射组和照射组，共8个实验组。利用Western blotting法检测各组细胞Smac蛋白的表达，利用流式细胞术检测细胞凋亡率和不同周期进程细胞百分率。结果：Western blotting法检测，经条件复制型腺病毒CRAd.pE-Smac感染、乏氧和照射后，Smac蛋白表达水平增加，CARd.pE-Smac+乏氧+4Gy组Smac蛋白表达水平最高。流式细胞术检测，与对照组比较，CARd.pE-Smac组、乏氧组和CARd.pE-Smac+乏氧组细胞凋亡率均明显升高（P<0.05或P<0.01）；与相应未照射组比较，4 Gy照射后CARd.pE-Smac+4Gy组、乏氧+4Gy组和CARd.pE-Smac+乏氧+4Gy组细胞凋亡率均明显升高（P<0.05或P<0.01），CARd.pE-Smac+乏氧+4Gy组升高最为明显，且S期和G₂/M期细胞百分率明显增加（P<0.05或P<0.01），与诱导凋亡的结果有相似的趋势。结论：在条件复制型腺病毒CRAd.pE-Smac感染MDA-MB-231细胞、并经乏氧和辐射处理后，实现了三重靶向介导的Smac过表达，其具有促进肿瘤细胞凋亡和诱导G₂/M期细胞阻滞的作用。

Effects of Smac overexpression mediated by triple-targeting on apoptosis and cell cycle progression of breast cancer MDA-MB-231 cells

Objective:To obtain the conditionally replicative adenovirus with triple-targeting Smac overexpression using gene recombination technology,and to explore its effects on the apoptosis and cell cycle progression of MDA-MB-231 cells.Methods:The triple-targeting Smac overexpression vector pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K was constructed by gene recombination technology,which was recombined with the skeleton vector pAdEasy in the BJ5183 bacteria (AdEasy-1+-) to obtain the conditionally replicative adenovirus CRAd.pE-Smac.After the MDA-MB-231 cells were infected with CRAd.pE-Smac,the cancer cells were mimiced into hypoxic status with chemical reagent CoCl2,then control group,CRAd.pE-Smac group,hypoxia group and CRAd.pE-Smac+-hypoxia group were set up;the cells were irradiated with 4 Gy X-rays,and each group was divided into nonirradiation group and irradiation group.The Smac protein expression was detected by Western blotting assay,the apoptotic rates and the percentages of cells at different phases were detected by flow cytometry.Results:The Western blotting results showed that the Smac protein expressions were increased after infection of CRAd.pE-Smac,hypoxia and 4 Gy irradiation,especially in CRAd.pE-Smac +hypoxia+4 Gy irradiation group.The FCM results showed that the apoptotic rates in CARd.pE-Smac,hypoxia,CARd.pE-Smac + hypoxia group were increased compared with control group (P＜0.05 or P＜0.01),and the apoptotic rates of cells irradiated with 4 Gy were significantly increased compared with the unirradiated cells (P＜0.05 or P＜0.01),especially in CRAd.pE-Smac + hypoxia + 4 Gy irradiation group;the percentages of the cells at S and G2/M phases in irradiation groups were significantly increased (P＜ 0.05 or P＜0.01),which had the similar regularity with the apoptotic change.Conclusion:After the MDA-MB-231 cells are infected with the conditionally replicative adenovirus CRAd.pE-Smac and treated with hypoxia and irradiation,the triple-targeting Smac overexpression can be achieved,and it has the role of promoting the cancer cell apoptosis and inducing the G2/M arrest.