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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。
OBJECTIVE: To observe the effect of double gene TRAIL and endostatin carried by recombinant plasmid pshuttle-Egr1-shTRAIL-shES combined with X-ray irradiation on the proliferation, cycle and apoptosis of ECV304 vascular endothelial cells. Methods: The experiment was divided into control group, empty vector pshuttle transfection group, TRAIL single gene recombinant plasmid pshuttle-Egr1-shTRAIL transfection group, endostatin single gene recombinant plasmid pshuttle-Egr1-shES transfection group and TRAIL, endostatin double gene recombinant plasmid pshuttle-Egr1-shTRAIL-shES transfection group. Cell transfection was performed using liposome-mediated methods without control transfection. After transfection, the cells were irradiated with X-rays (irradiation doses of 0, 0.1, 0.5, 1.0, 2.0 and 5.0 Gy respectively). The expressions of TRAIL and endostatin in transfected cells were detected by ELISA. MTT and PI single staining Or / and AnnexinⅤ double staining flow cytometry (FCM) TRAIL, endostatin single / double gene therapy combined with radiotherapy on ECV304 cell proliferation, cell cycle and apoptosis. Results: The expression of TRAIL and endostatin in ECV304 cells transfected with pshuttle-Egr1-shTRAIL-shES at each time point was significantly higher than that at 0h (P <0.01) (P <0.05 or P <0.01). The expression of TRAIL and endostatin was induced by different doses of X-ray irradiation. The protein expression levels of TRAIL and endostatin were significantly increased with the increase of irradiation dosage (P <0.05 or P <0.01). MTT results showed that A490 values of ECV304 cells in pshuttle-Egr1-shTRAIL, pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups were significantly lower than those in control group and pshuttle group after X-ray irradiation and showed a certain time- And dose-effect relationship, accompanied with a significant increase in the rate of apoptosis, G2 + M phase cells significantly increased percentage and G0 / G1 phase cells decreased significantly. The above-mentioned cellular effects, especially in the pshuttle-Egr1-shTRAIL-shES group, were significantly different from those in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shES groups (P <0.05 or P <0.01). Conclusion: Dual gene therapy with TRAIL and endostatin combined with radiotherapy can inhibit the growth of ECV304 cells, affect the cell cycle progression and promote apoptosis. The combination of TRAIL and endostatin is superior to radiotherapy alone or TRAIL / endostatin single gene radiotherapy.