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

作　　者：毛富刚[1] 武馨馨 陈鑫昊 李思 严丹[4] 肖志原 贺继刚[3] Mao Fugang;Wu Xinxin;Chen Xinhao;Li Si;Yan Dan;Xiao Zhiyuan;He Jigang(Department of Ultrasound,Yunnan First People's Hospital,Kunming 650000,China;Yunnan University of Traditional Chinese Medicine,Kunming 650032,China;Department of Cardiovascular Surgery,Yunnan First People's Hospital,Kunming 650000,China;Department of Internal Medicine and Critical Care Medicine,Yunnan First People's Hospital,Kunming 650000,China)

机构地区：[1]云南省第一人民医院超声科,昆明650000 [2]云南中医药大学,昆明650032 [3]云南省第一人民医院心脏大血管外科,昆明650000 [4]云南省第一人民医院内科重症医学科,昆明650000

出　　处：《中国综合临床》2024年第3期191-196,共6页Clinical Medicine of China

基　　金：国家自然科学基金(82060299);云南省卫生健康委员会医学学科带头人项目(D-2019020);云南省“万人计划”-青年拔尖人才(KH-SWR-QNBJ-2019-002);云南省第一人民医院临床医学中心开放项目(2021LCZXXF-XZ04,2022LCZXKF-HX05);云南省科技厅科技计划项目(202101AY070001-034,202101AY070001-272);云南省2023年本科教育教学改革研究项目(2023BKXJJG-F04002);云南省“兴滇英才支持计划”(XDYC-MY-2022-0037)。

摘　　要：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.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)+

关 键 词：骨髓间充质干细胞 NKX2.5 抗凋亡 心肌梗死 

分 类 号：R542.22[医药卫生—心血管疾病]