辐射诱导的重组载体pEgr1-hsTRAIL对肺腺癌A549细胞的生长抑制作用

目的：构建Egr1介导的人分泌型肿瘤坏死因子相关的凋亡诱导配体（hsTRAIL）重组表达载体pEgr1-hsTRAIL，探讨其对人肺腺癌A549细胞的生长抑制作用。方法：利用基因重组技术构建Egr1介导的hsTRAIL重组载体，PCR、酶切和测序鉴定正确后转染A549细胞，给予6 Gy X射线照射。实验分为对照(Control)， pEgr1-hsTRAIL、6 Gy X射线和pEgr1-hsTRAIL+6 Gy X射线组。ELISA法检测各组A549细胞中hsTRAIL的表达， MTT法检测细胞增殖能力，流式细胞术检测细胞周期， TUNEL法检测细胞凋亡变化。结果：成功构建Egr1调控的hsTRAIL重组载体pEgr1-hsTRAIL，质粒转染后经6 Gy X射线照射，对照组、6 Gy组和pEgr1-hsTRAIL组hsTRAIL蛋白表达随时间延长变化不明显，而pEgr1-hsTRAIL+ 6 Gy组随时间延长hsTRAIL蛋白表达显著增加（P<0.05或P<0.01），8 h达到峰值；pEgr1-hsTRAIL组A549细胞增殖能力与对照组比较无明显差异，6 Gy和pEgr1-hsTRAIL+ 6 Gy组A549细胞增殖能力较对照组显著降低，pEgr1-hsTRAIL+ 6 Gy组降低更明显（P<0.05或P<0.01）；与对照组比较，pEgr1-hsTRAIL组A549细胞各期百分比变化不明显，而6 Gy和pEgr1-hsTRAIL+ 6 Gy组G0/G1期细胞百分比明显增加（P<0.05），G2/M期细胞百分比明显降低（P<0.05），S期细胞百分比无明显变化；各期细胞百分比在6 Gy和pEgr1-hsTRAIL+ 6 Gy组基本一致；与对照组比较，pEgr1-hsTRAIL组A549细胞凋亡百分比无明显变化，而6 Gy和pEgr1-hsTRAIL + 6 Gy组A549细胞凋亡百分比明显增加（P<0.01），其中pEgr1-hsTRAIL+ 6 Gy组增加更明显。结论：成功构建Egr1介导的hsTRAIL重组表达载体pEgr1-hsTRAIL，其能够增加辐射对A549细胞的增殖抑制和凋亡诱导作用，而对细胞周期分布影响不大。

Inhibitory effect of recombinant vetor pEgr1-hsTRAIL induced by radiation on growth of lung adenoxarcinoma A549 cells

Objective To construct human secreted TRAIL(hsTRAIL) recombinant vetor pEgr1-hsTRAIL mediated by Egr1,and to explore the inhibitory effect on the growth of lung adenocarcinoma A549 cells.Methods The hsTRAIL vetor mediated by Egr1 was constructed by gene recombination techinique,the A549 cells were transfected with the plasmid after identification by PCR,restrictive enzyme digestion and sequencing,and irradiated by 6 Gy X-rays.There were control group,pEgr1-hsTRAIL group,6 Gy X-rays group and pEgr1-hsTRAIL + 6 Gy X-rays group in the experiment.The expression of hsTRAIL in A549 cells was detected by ELISA method,the cell proliferation was detected by MTT assay,the cycle changes of cell cycle were detected by flow cytometry and the apoptosis was measured by TUNEL method.Results The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 was constructed successfully.The cells were irradiated by 6 Gy X-rays after transfected with plasmid.The hsTRAIL protein expressions in control,6 Gy and pEgr1-hsTRAIL groups didn’t change significantly with the time prolongation,but the expression in pEgr1-hsTRAIL+6 Gy group was increased significantly with the time prolongation(P<0.05 or P<0.01),and reached to peak value at 8 h.There was no significant difference of A549 cell proliferation ability between control group and pEgr1-hsTRAIL group,but the proliferation abilities in 6 Gy and pEgr1-hsTRAIL+6 Gy groups were decreased significantly compared with control group,especially in pEgr1-hsTRAIL+6 Gy group(P<0.05 or P<0.01).Compared with control group,the percentages of A549 cells at different phases in pEgr1-hsTRAIL group didn’t change significantly,but the percentages of A549 cells at G0/G1 phase in 6 Gy and pEgr1-hsTRAIL+ 6 Gy groups were increased significantly(P<0.05),the percentages of A549 cells at G2/M phase were decreased significantly(P<0.05),the percentages of A549 cells at S phase didn’t change significantly.The percentages of A549 cells at different phases in 6 Gy group were basically consistent with those in pEgr1-hsTRAIL+6 Gy group.Compared with control group,the apoptotic percentage of A549 cells in pEgr1-hsTRAIL group had no significant change,but they were increased significantly in 6 Gy and pEgr1-hsTRAIL+6 Gy groups compared with control group(P<0.01),especially in pEgr1-hsTRAIL+6 Gy group.Conclusion The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 is successfully constructed,the vector can enhance the inhibitory effects and effect of inducing apoptosis of radiation on the A549 cells,but it affects cell distribution little.