摘要目的 探讨9型腺相关病毒介导血小板源性生长因子-B(PDGF-B)基因转染修饰大鼠心肌细胞的可行性、安全性及其抗凋亡功能.方法 将携带PDGF-B基因的9型腺相关病毒(rAAV9-PDGF-B)转染大鼠心肌细胞(H9C2),收集经转染后不同时间点(1、3、5、14、21、28 d)心肌细胞,提取蛋白质进行Western印迹测定,观察PDGF-B表达情况.细胞免疫荧光法检测rAAV9的转染效率,AlamarBlue法对rAAV9-PDGF-B转染心肌细胞进行安全性评估,并通过双氧水(H2O2)诱导凋亡,检测高表达PDGF-B的心肌细胞的凋亡相关蛋白及Tunel阳性率.结果 rAAV9-PDGF-B转染心肌细胞后第3天开始,PDGF-B表达上调,随后表达逐渐增强,到第5天蛋白持续平稳表达,持续表达至28 d.转染后第5天,表达PDGF-B的心肌细胞约为78.6％.不同时间点的心肌细胞的还原比率均接近于1.0.双氧水诱导凋亡后,可下调高表达PDGF-B心肌细胞的Bax及Tunel阳性率,同时上调Bcl-2及P-Akt的表达,与PDGF-B蛋白抗凋亡组相比差异无统计学意义(P＞0.05).结论 rAAV9-PDGF-B能成功转染大鼠心肌细胞,使心肌细胞持续高表达PDGF-B达28 d以上,对心肌细胞无明显不良反应.高表达PDGF-B的心肌细胞同样具有抗凋亡的功能.

abstractsObjective To explore the feasibility,safety and anti-apoptosis capacity of transfection of cardiomyocytes in rats using recombinant adeno-associated virus-9 containing platelet-derived growth factor-B genes (rAAV9-PDGF-B).Methods Rat cardiomyocytes (H9C2) were transfected with rAAV9-PDGF-B and harvested at different time points (days 1,3,5,14,21 and 28),then the protein was ertracted to detect the expression of PDGF-B by Western Blotting.The cell immunofluorescence assay was employed to measure the transfection efficiency,and Alamar Blue assay was applied to evaluate the safety of transfection with rAAV9-PDGF-B.The apoptosis-related proteins and the ratio of Tunel-positive in the cardiomyocytes overexpressing PDGF-B were assayed following H2O2-induced apoptosis.Results Following transfection with rAAV9-PDGF-B,the cardiomyocytes overexpressed PDGF-B at day 3.This was followed by a gradual increase that reached to a plateau at day 5 and lasted for 28 days.There was approximately 78.6％ of cardiomyocytes expressing PDGF-B at day 5.The reduction ratio of cardiomyocytes at different time points approached to 1.0.Moreover,H2O2-induced apoptosis was accompanied by the down-regulation of Bax and Tunel-positive cells and up-regulation of Bcl-2 and P-Akt expression,which did not evidence significant difference with the anti-apoptosis group (P＞0.05).Conclusion The rAAV9-PDGF-B can effectively transfect cardiomyocytes that persistently expresses PDGF-B for 28 days.No significant toxic effects on cardiomyocytes is found.The cardiomyocytes overexpressing PDGF-B also have the capacity of protecting cells against H2O2-induced apoptosis.