干扰Nrf2基因后骨髓间充质干细胞移植对大鼠心肌梗死后心肌凋亡的影响

Effect of silencing Nrf2 gene on myocardial apoptosis after rat myocardial infarction with bone marrow mesenchymal stem cell transplantation

目的观察干扰核因子相关因子2(Nrf2)的骨髓间充质干细胞(mesenchymal stem cells,MSCs)注射大鼠心肌梗死局部后细胞凋亡情况及对心功能的影响。方法建立心肌梗死大鼠模型,随机分为:1干扰Nrf2基因的MSCs移植(Nrf2干扰组)组;2空载慢病毒修饰的MSCs移植(Nrf2正常组);3生理盐水注射对照组(对照组,n=12)。心肌梗死细胞移植后7 d,细胞免疫荧光法检测心肌局部caspase3蛋白的表达;移植后28 d HE染色评价梗死后心肌组织病理变化,TTC染色评估梗死面积;采用蛋白印迹法(Western blot)检测心肌梗死区Nrf2和凋亡蛋白Bcl2、Bax的蛋白表达水平。结果心梗细胞移植后7d,心肌梗死区域有EGFP标记的MSCs,部分EGFP表达位置检测到Caspase3表达阳性,Nrf2干扰组凋亡蛋白Caspase3的荧光强度强,Nrf2正常组Caspase3的荧光强度弱,对照组无EGFP表达;细胞移植后28 d,HE染色结果显示,与Nrf2正常组比较,Nrf2干扰组心肌组织肌纤维排列紊乱,炎性细胞浸润明显,而Nrf2正常组细胞排列整齐,炎性细胞浸润减少;心肌TTC染色结果显示,Nrf2干扰组的心肌梗死面积为(35.30±0.41)%,较Nrf2正常组[(25.07±0.15)%]明显增加(P<0.05),与对照组[(35.40±0.70)%]比较无差异(P>0.05);Western blot结果显示,与Nrf2正常组比较,Nrf2干扰组心肌中Nrf2蛋白和抗凋亡蛋白Bcl-2表达下降,而促凋亡蛋白Bax表达增加,差异有统计学意义(P<0.05),Nrf2干扰组与对照组比较,各蛋白表达无差异性(P>0.05);心功能改善方面,Nrf2干扰组EF[(40.83±2.67)%]较Nrf2正常组[(52.35±1.58)%]降低(P<0.050而心室容积较[(LVDd,(1.090±0.061),LVDs(0.750±0.062)]较Nrf2正常组[(LVDd,(0.790 0±0.024 5),LVDs(0.610±0.026)]增加。结论移植干扰Nrf2的MSCs后,心肌梗死区凋亡相关蛋白表达增加,降低了外源性MSCs对梗死心脏的修复能力。

Objective To determine the effect of transplantation of bone marrow mesenchymal stem cells（ MSCs） after RNA-interfered nuclear factor erythroid 2-related factor 2（ Nrf2） on myocardial apoptosis and cardiac function in rats after myocardial infarction（ MI）. Methods The established MI rat models were randomly divided into MSCs transplantation group with Nrf2 gene silence（ Nrf2 interference group,n = 12）,MSCs transplantation group with no-load lentivirus modification（ Nrf2 normal group,n = 12）,and normal saline injection group（ control group,n = 12）. On the 7th day after cell transplantation,the expression of caspase 3 in myocardial tissue was observed with cell immunofluorescence assay. On the 28 th day after cell transplantation,pathological changes in myocardial tissues after infarction was evaluated after HE staining,the infarct size was evaluated with TTC staining,the protein expression of Nrf2,Bcl2 and Bax in MI region was detected by Western blotting. Results On the 7th day after cell transplantation,the immunofluorescence results showed that the fluorescence intensity of protein caspase 3 in myocardial tissue was significantly increased in MSCsNrf2- /-group than MSCsNrf2 ＋ /-group,and no fluorescence was observed in the control group.On the 28 th day after cells transplantation,HE staining results showed that there were obvious myocardial tissue muscle fibers in disorganized arrangement and filaments breakage and infiltration of inflammatory cells in the Nrf2 interference group but the myocardial tissue muscle fibers were well-arranged and less infiltration of inflammatory cells were seen in the Nrf2 normal group. Myocardial TTC staining showed that the myocardial infarct size was（ 35. 30 ± 0. 41） % in the Nrf2 interference group,which was significantly higher than that in the Nrf2 normal group [（ 25. 07 ± 0. 15） %,P〈0. 05],but had no significant difference with the control group[（ 35. 40 ± 0. 70） %,P〈0. 05]. Western blot results showed that the expression levels of Nrf2 and antiapoptotic protein Bcl-2 were decreased,while that of pro-apoptotic protein Bax was increased in the Nrf2 interference group than the Nrf2 normal group（ P〈0. 05）,but no difference was found in the expression of above molecules between the former group and control（ P〈0. 05）. The Nrf2 interference group had obviously lower ejection fraction（ EF） and larger ventricular volumethan the Nrf2 normal group [EF：（ 40. 83 ± 2. 67） %vs（ 52. 35 ± 1. 58） %,P〈0. 05; left ventricular end diastolic diameter： 1. 090 ± 0. 061 vs 0. 790 0 ± 0. 024 5,left ventricular end systolic diameter： 0. 750 ± 0. 062 vs 0. 610 ± 0. 026]. Conclusion Transplantation of Nrf2-interfered MSCs suppresses the expression of anti-apoptotic protein while promotes that of pro-apoptotic protein in myocardial infarction zone,and reduces the repair ability of exogenous MSCs for infarcted heart.