凋亡诱导因子基因敲减对骨髓间充质干细胞移植治疗心肌梗死的影响

Effect of apoptosis-inducing factor gene knockdown on bone marrow mesenchymal stem cell transplantation for myocardial infarction

背景:众多基础与临床试验证实:骨髓间充质干细胞移植后的低存活率严重制约着其发挥长期的治疗效果。课题组前期研究发现凋亡相关因子在骨髓间充质干细胞凋亡过程中发挥了重要作用,其中凋亡诱导因子(apoptosis-inducing factor,AIF)蛋白可能是其中一个关键因子。目的:将AIF敲减的骨髓间充质干细胞移植至小鼠梗死心肌中,验证低AIF表达骨髓间充质干细胞移植后的存活情况以及对进一步改善心功能的重要性。方法:首先,通过LV-AIF-shRNA慢病毒感染骨髓间充质干细胞下调AIF蛋白表达,应用流式细胞术及Western blot、RT-qPCR检测慢病毒的感染效率,CCK-8检测AIF敲减骨髓间充质干细胞在缺血缺氧条件下的细胞活力;然后,构建急性心肌梗死小鼠模型,分别将正常及AIF敲减的骨髓间充质干细胞移植至心肌梗死区域,免疫荧光检测AIF蛋白的表达,ELISA检测血清脑钠尿肽水平,心脏超声检测心功能,Masson染色观察心肌纤维化情况,RT-qPCR检测AIF敲减骨髓间充质干细胞移植后SRY基因表达反映细胞存活情况。结果与结论:①LV-AIF-shRNA慢病毒感染成功构建AIF基因敲减的骨髓间充质干细胞,感染效率为97.7%,且AIF表达有显著下降(P<0.001);②在缺血缺氧培养状态下,AIF基因敲减骨髓间充质干细胞较正常骨髓间充质干细胞的细胞活力显著增加;③与移植正常骨髓间充质干细胞相比,AIF基因敲减骨髓间充质干细胞移植后在梗死心肌中的存活数目显著升高至3.71倍(P<0.001),并且显著减少了梗死区域AIF蛋白表达、心肌纤维化程度;④与移植正常骨髓间充质干细胞相比,AIF基因敲减骨髓间充质干细胞移植后血清脑钠尿肽水平显著降低(P<0.05),左室射血分数、左室缩短分数均有显著改善(P<0.05);⑤结果表明:AIF基因敲减可通过增强骨髓间充质干细胞移植后的细胞活力、增加骨髓间充质干细胞在供体内的存活从而减少心肌纤维化,改善急性心肌梗死后心功能。

BACKGROUND:Numerous basic and clinical trials have confirmed that the low survival rate after bone marrow mesenchymal stem cell transplantation is a serious constraint on its long-term therapeutic effect.Previous studies have shown that apoptosis-related factors play an important role in the apoptosis of bone marrow mesenchymal stem cells,of which apoptosis-inducing factor may be a key factor.OBJECTIVE:Bone marrow mesenchymal stem cells,of which apoptosis-inducing factor was knocked down,were transplanted into infarcted myocardium of mice,aiming to certify the importance of apoptosis-inducing factor in the survival of bone marrow mesenchymal stem cells to further recover cardiac function after infarction.METHODS:Firstly,bone marrow mesenchymal stem cells were infected with LV-AIF-shRNA lentivirus to down-regulate the expression of apoptosis-inducing factor protein.Flow cytometry,western blot assay,and RT-qPCR were used to detect the infection efficiency of lentivirus.CCK-8 assay was used to detect the cell viability of bone marrow mesenchymal stem cells with apoptosis-inducing factor knockdown under hypoxic and ischemic conditions.Then,with the mouse model of acute myocardial infarction constructed,the normal bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells with apoptosisinducing factor gene knockdown were transplanted into the model,respectively.The expression of apoptosis-inducing factor was examined by fluorescence immunoassay.Serum brain natriuretic peptide levels were detected by ELISA.Cardiac ultrasound was used to detect cardiac function.Myocardial fibrosis was observed by Masson staining.The expression of SRY gene was detected by RT-qPCR in apoptosis-inducing factor-knocked bone marrow mesenchymal stem cells after transplantation,reflecting cell survival.RESULTS AND CONCLUSION:(1)Bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were successfully established by LVAIF-shRNA lentivirus infection,following 97.7%of infection efficiency,and notably decline of the expression of apoptosis-inducing factor(P<0.001).(2)Under ischemia and hypoxia,the cell viability of apoptosis-inducing factor knockdown bone marrow mesenchymal stem cells was significantly increased compared with normal bone marrow mesenchymal stem cells.(3)Compared with normal bone marrow mesenchymal stem cells after transplantation,the survival number of bone marrow mesenchymal stem cells in the infarcted myocardium after apoptosis-inducing factor gene knockdown was significantly increased to 3.71 times(P<0.001),and the apoptosis-inducing factor protein expression and myocardial fibrosis degree in the infarcted area were significantly reduced.(4)Compared with normal bone marrow mesenchymal stem cells,the serum brain natriuretic peptide level of bone marrow stem cells with apoptosis-inducing factor gene knockdown after transplantation was significantly decreased(P<0.05),and left ventricular ejection fraction and left ventricular shortening fraction were significantly improved(P<0.05).(5)These findings confirm that apoptosis-inducing factor gene knockdown can reduce myocardial fibrosis and improve cardiac function after acute myocardial infarction via enhancing the bone marrow mesenchymal stem cell viability and increasing the bone marrow mesenchymal stem cell survival after transplantation in the donor.