CAJ | 학술논문

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.
목적 탐토9형선상관병독개도혈소판원성생장인자-B(PDGF-B)기인전염수식대서심기세포적가행성、안전성급기항조망공능.방법 장휴대PDGF-B기인적9형선상관병독(rAAV9-PDGF-B)전염대서심기세포(H9C2),수집경전염후불동시간점(1、3、5、14、21、28 d)심기세포,제취단백질진행Western인적측정,관찰PDGF-B표체정황.세포면역형광법검측rAAV9적전염효솔,AlamarBlue법대rAAV9-PDGF-B전염심기세포진행안전성평고,병통과쌍양수(H2O2)유도조망,검측고표체PDGF-B적심기세포적조망상관단백급Tunel양성솔.결과 rAAV9-PDGF-B전염심기세포후제3천개시,PDGF-B표체상조,수후표체축점증강,도제5천단백지속평은표체,지속표체지28 d.전염후제5천,표체PDGF-B적심기세포약위78.6％.불동시간점적심기세포적환원비솔균접근우1.0.쌍양수유도조망후,가하조고표체PDGF-B심기세포적Bax급Tunel양성솔,동시상조Bcl-2급P-Akt적표체,여PDGF-B단백항조망조상비차이무통계학의의(P＞0.05).결론 rAAV9-PDGF-B능성공전염대서심기세포,사심기세포지속고표체PDGF-B체28 d이상,대심기세포무명현불량반응.고표체PDGF-B적심기세포동양구유항조망적공능.
Objective 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.