【摘要】 目的:观察重组质粒pshuttle-Egr1-shTRAIL-shES携带的双基因TRAIL和endostatin联合X射线照射后,对血管内皮细胞ECV304增殖、周期和凋亡的影响。方法:实验分为对照组、空载体pshuttle转染组、TRAIL单基因重组质粒pshuttle-Egr1-shTRAIL转染组、endostatin单基因重组质粒pshuttle-Egr1-shES转染组和TRAIL、endostatin双基因重组质粒pshuttle-Egr1-shTRAIL-shES转染组。细胞转染采用脂质体介导的方法进行,对照组不转染。细胞转染后给予X射线照射(照射剂量分别为0、0.1、0.5、1.0、2.0和5.0Gy),采用ELISA法检测转染细胞中TRAIL和endostatin蛋白的表达,并分别采用MTT及PI单染或/和AnnexinⅤ双染流式细胞术(FCM)检测TRAIL、endostatin单/双基因治疗联合放射治疗对ECV304细胞增殖、细胞周期和凋亡的影响。结果:2.0Gy X射线照射后与0h比较,各时间点转染pshuttle-Egr1-shTRAIL-shES的ECV304细胞上清中TRAIL和endostatin蛋白表达水平明显升高(P<0.01),分别于12和24h达峰值;不同剂量X射线照射可诱导TRAIL和endostatin蛋白表达,且蛋白表达水平随照射剂量的增加而明显升高(P<0.05或P<0.01)。MTT结果显示,X射线照射后,pshuttle-Egr1-shTRAIL、pshuttle-Egr1-shES和pshuttle-Egr1-shTRAIL-shES组ECV304细胞A490值均明显低于对照组和pshuttle组,并显示一定的时间-效应和剂量-效应关系,并伴有细胞凋亡率明显增加、G2+M期细胞百分数明显上升和G0/G1期细胞百分数明显下降。上述细胞效应,尤以pshuttle-Egr1-shTRAIL-shES组变化最为明显,与pshuttle-Egr1-shTRAIL和pshuttle-Egr1-shES组比较差异有统计学意义(P<0.05或P<0.01)。结论:TRAIL和endostatin双基因联合放射治疗可抑制ECV304细胞生长,影响细胞周期进程,促进细胞凋亡,且其治疗效果优于单纯放射治疗或TRAIL/endostatin单基因-放射治疗。更多还原

【Abstract】 Objective To study the effects of TRAIL and endostatin double genes in recombinant plasmid pshuttle-Egr1-shTRAIL-shES combined with X-ray irradiation on the proliferation,cell cycle procession and apoptosis in human vascular endothelial ECV304 cells.Methods Cells were divided into control,empty vector pshuttle,TRAIL single-gene plasmid pshuttle-Egr1-shTRAIL,endostatin single-gene plasmid pshuttle-Egr1-shES and TRAIL plus endostatin double-gene plasmid pshuttle-Egr1-shTRAIL-shES transfection groups.The cell transfection was done by lipofectamine-mediated method,and no transfection in control group.After plasmid transfection,the cells were exposed under X-ray irradiation at the doses of 0,0.1,0.5,1.0,2.0 and 5.0 Gy,respectively.Then the time-course and dose-effect patterns of the TRAIL and endostatin protein expressions induced by irradiation were detected by ELISA,and the effects of TRAIL and/or endostatin single or double gene therapy combined with radiotherapy on the proliferation,cell cycle procession and apoptosis in ECV304 cells were detected by MTT assay,flow cytometry(FCM) with PI single-staining or/and Annexin Ⅴ double-staining.Results After 2.0 Gy X-ray irradiation,the expression levels of TRAIL and endostatin proteins in the supernatant of cultured ECV304 cells transfected with pshuttle-Egr1-shTRAIL-shES at other time-points were increased when compared with those at 0 h(P<0.01),and reached to the peak value at 24 and 12 h,respectively.Furthermore,their expression levels of TRAIL and endostatin protein induced by irradiation were increased significantly with the enlargement of radiation doses(P<0.05 or P<0.01).Under the exposure of X-ray irradiation,as compared with those in control and pshuttle groups,the A490 values detected with MTT in pshuttle-Egr1-shTRAIL,pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups were reduced in a time-and dose-dependent manner,and the apoptotic rates and the cell percentages in G2 + M phase were increased,while those in G0/G1 phase were declined.In particular,the cellular effects mentioned above in pshuttle-Egr1-shTRAIL-shES group were significantly higher than those in pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shES groups(P<0.05 or P<0.01).Conclusion TRAIL and endostatin double-gene-therapy in combination with radiotherapy could inhibit cell proliferation,affect cell cycle procession and promote apoptosis in ECV304 cells,and its therapeutic efficacy is better than those of single gene-radiotherapy or radiotherapy.更多还原