过表达NKx2.5基因对间充质干细胞抗凋亡能力及对心肌梗死后心功能的影响

Overexpression of NKx2.5 gene affects the anti apoptotic ability of mesenchymal stem cells and cardiac function after myocardial infarction

目的探讨过表达Nkx2.5基因对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)抗细胞凋亡能力及对心肌梗死后心功能的影响。方法采用糖氧剥夺[低氧无血清(oxygen glucose deprivation/reoxygenat,OGD/R)]条件下培养,建立细胞缺血模型,实验分为4组:正常条件培养下的骨髓间充质干细胞组(以下简写为BMSC组)、糖氧剥夺条件下培养的BMSC组(以下简写为BMSC+OGD/R组)、糖氧剥夺条件下培养的过表达空载体BMSC组(以下简写为BMSC空载体+OGD/R组)、糖氧剥夺条件下培养的过表达Nkx2.5 BMSC组(以下简写为BMSC^(Nkx2.5)+OGD/R组)。采用流式细胞技术检测各组BMSC的凋亡率,并提取BMSC蛋白,采用Western Blot法检测各组BMSC中的Caspase-3及前体、Caspase-8及前体、Caspase-9及细胞色素C蛋白量及Nkx2.5的表达,明确体外抗凋亡的途径。采用结扎冠状动脉左前降支建立小鼠心肌梗死模型,实验分为五组:假手术组、心肌梗死未处理组、心肌梗死后BMSC组、心肌梗死后BMSC^(空载体)组、心肌梗死后BMSC^(NKx2.5)组。通过心脏彩超评估小鼠心功能变化。正态分布的计量资料组间比较采用方差分析,两两比较采用LSD-t检验。结果BMSC+OGD/R组细胞凋亡率(12.98±1.24)%高于BMSC组(7.82±0.42)%,差异有统计学意义(P<0.001);BMSC^(NKx2.5)+OGD/R组细胞凋亡率(11.26±0.22)%低于BMSC+OGD/R组(12.98±1.24)%、BMSC^(空载体)+OGD/R组(13.14±0.70)%,差异有统计学意义(P<0.05)。BMSC^(Nkx2.5)+OGD/R组Caspase-3(0.72±0.08)及前体(0.89±0.09)、Caspase-8(0.63±0.08)及前体(0.85±0.12)、Caspase-9(0.87±0.09)、细胞色素C(0.91±0.10)、Nkx2.5(1.54±0.16)的表达与BMSC组[(0.36±0.08)、(1.13±0.04)、(0.36±0.06)、(1.12±0.13)、(1.23±0.08)、(0.60±0.05)、(0.67±0.14)]、BMSC+OGD/R组[(1.05±0.10)、(0.62±0.04)、(1.07±0.09)、(0.57±0.07)、(0.55±0.08)、(1.25±0.09)、(0.71±0.04)]、BMSC^(空载体)+OGD/R组[(1.16±0.16)、(0.64±0.06)、(1.19±0.16)、(0.56±0.06)、(0.50±0.06)、(1.28±0.06)、(0.73±0.04)]表达比较,差异有统计学意义(均P<0.05)。体内实验发现经BMSC^(Nkx2.5)组处理的小鼠心肌梗死后心脏射血分数(29.05±7.07)%较心肌梗死后BMSC组(16.57±2.09)%、心肌梗死后BMSC^(空载体)组(18.08±3.27)%改善明显(均P<0.05)。结论BMSC^(Nkx2.5)可能通过抑制死亡受体通路及线粒体信号通路增强BMSC抗凋亡能力,改善心肌梗死后心功能。

Objective To investigate the effects of overexpression of Nkx2.5 gene on the anti apoptotic ability of bone marrow mesenchymal stem cells(BMSCs)and cardiac function after myocardial infarction.Methods A cell ischemia model was established by culturing cells under oxygen glucose deprivation/reoxygenat(OGD/R)conditions.The experiment was divided into four groups:bone marrow mesenchymal stem cells cultured under normal conditions(BMSC group),BMSC group cultured under glucose and oxygen deprivation(BMSC+OGD/R group),overexpressed empty vector BMSC group cultured under glucose and oxygen deprivation(BMSC^(NC)+OGD/R group),and overexpressed Nkx2.5 BMSC group cultured under glucose and oxygen deprivation(BMSC^(Nkx2.5)+OGD/R group).The apoptosis rate of BMSCs in each group was detected via flow cytometry,and BMSC protein was extracted.The expression of caspase-3 and pro-caspase-3,caspase-8 and pro-caspase-8,caspase-9,and cytochrome C protein and expression of Nkx2.5 in the BMSCs of each group were detected by Western blot to determine the anti-apoptotic pathway in vitro.The model of myocardial infarction in mice was established by ligating the left anterior descending branch of coronary artery.The experiment was divided into five groups:sham surgery group,myocardial infarction untreated group,myocardial infarction tail vein injection of BMSC group,myocardial infarction tail vein injection of BMSC empty body group,myocardial infarction tail vein injection of BMSC overexpression Nkx2.5 group.The changes of cardiac function in mice were evaluated by echocardiography.Normal distribution econometric data were compared between groups using convenient analysis,and pairwise comparisons were conducted using LSD-t test.Results The apoptosis rate of the BMSC+OGD/R group(12.98±1.24)% was higher than that of the BMSC group(7.82±0.42)%,and the difference was statistically significant(P<0.001).The apoptosis rate of the BMSC^(NKx2.5)+OGD/R group(11.26±0.22)% was lower than that of the BMSC+OGD/R group(12.98±1.24)% and the BMSC^(NC)+OGD/R group(13.14±0.70)%,with statistically significant differences(P<0.05).Compared to BMSC group((0.36±0.08),(1.13±0.04),(0.36±0.06),(1.12±0.13),(1.23±0.08),(0.60±0.05),(0.67±0.14)),BMSC+OGD/R group((1.05±0.10),(0.62±0.04),(1.07±0.09),(0.57±0.07),(0.55±0.08),(1.25±0.09),(0.71±0.04))and BMSC^(NC)+OGD/R group((1.16±0.16),(0.64±0.06),(1.19±0.16),(0.56±0.06),(0.50±0.06),(1.28±0.06),(0.73±0.04)),the expression of Caspase-3(0.72±0.08)and pro-caspase-3(0.89±0.09),Caspase-8(0.63±0.08)and pro-caspase-8(0.85±0.12),Caspase-9(0.87±0.09),cytochrome C(0.91±0.10),and Nkx2.5(1.54±0.16)in BMSC^(Nkx2.5)+OGD/R group was statistically significant(all P<0.05).In vivo experiments showed that the heart ejection fraction(29.05±7.07)% of mice treated with BMSC^(Nkx2.5) after myocardial infarction was significantly improved compared to the BMSC group(16.57±2.09)% and BMSC^(NC) group(18.08±3.27)%(all P<0.05).Conclusion BMSC^(Nkx2.5) may enhance the anti-apoptosis ability of BMSCs and improve cardiac function after myocardial infarction by inhibiting the death receptor pathway and the mitochondrial signal pathway.