论文部分内容阅读
目的评估外源性组织型纤溶酶原激活物(t-PA)对ECV304细胞中血管内皮细胞生长因子(VEGF)表达的影响。方法应用细菌内同源重组技术快速构建Adt-PA腺病毒重组质粒,转染不同病毒滴度Adt-PA至ECV304细胞,转染比率(MOI)分别为1∶10、1∶50、1∶100,选取合适MOI值;病毒转染后检测ECV304细胞内t-PA蛋白的表达。然后将培养的ECV304细胞分为三组,在培养液中加入Adt-PA混合培养24 h和48 h分别作为实验组1和实验组2,加入空病毒Ad混合培养48 h作为对照组,比较各组细胞中VEGF mRNA的转录和蛋白表达水平。结果成功构建了重组腺病毒Adt-PA。当MOI为1∶50时,细胞转染效率为(69.6±21.2)%,且对ECV304细胞增殖具有一定的促进作用;Adt-PA转染ECV304细胞后在72 h内随着时间的延长其蛋白表达量逐渐升高(P<0.01);细胞内VEGFmRNA的转录水平和蛋白表达量在实验组1和2中较对照组均有明显升高(P<0.01)。结论构建的t-PA腺病毒表达载体可有效感染ECV304细胞并可显著增加其VEGF的表达水平。
Objective To evaluate the effect of exogenous tissue plasminogen activator (t-PA) on the expression of vascular endothelial growth factor (VEGF) in ECV304 cells. Methods Adt-PA recombinant adenovirus was constructed by homologous recombination in bacteria and transfected into Adt-PA to ECV304 cells with different titer of transfection. The transfection ratio (MOI) was 1:10, 1:50, 1:100 respectively , Select the appropriate MOI value; detection of t-PA protein expression in ECV304 cells after transfection. The cultured ECV304 cells were divided into three groups. Adt-PA was added into the culture medium for 24 h and 48 h, respectively. The cells were divided into experimental group 1 and experimental group 2, and mixed with empty virus Ad for 48 h as control group VEGF mRNA transcription and protein expression in group cells. Results The recombinant adenovirus Adt-PA was successfully constructed. When the MOI was 1:50, the transfection efficiency was (69.6 ± 21.2)%, and had a certain promotion effect on the proliferation of ECV304 cells. The protein expression of ECV304 cells was increased with the prolongation of 72 hours after Adt-PA transfection (P <0.01). The transcriptional level and protein expression of VEGF mRNA in the experimental groups 1 and 2 were significantly higher than those in the control group (P <0.01). Conclusion The constructed t-PA adenovirus expression vector can effectively infect ECV304 cells and significantly increase the expression level of VEGF.