低氧预处理脂肪间充质干细胞移植对小鼠心肌梗死后心肌细胞凋亡及血管新生的影响

Effects of hypoxia-preconditioned adipose mesenchymal stem cells transplantation on apoptosis and angiogenesis in a mouse model of myocardial infarction

目的探讨低氧预处理脂肪间充质干细胞(ADSCs)移植对小鼠心肌梗死后细胞凋亡及新生血管形成的作用及机制。方法原代分离培养并鉴定小鼠ADSCs,取第3代ADSCs分为低氧组和常氧组分别置于低氧(2% O_2)和常氧(21%O2)环境中孵育6 d,观察低氧和常氧培养的ADSCs标志蛋白及Ki67表达,实时荧光定量PCR技术检测低氧和常氧培养ADSCs血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)、胎盘生长因子(PLGF)mRNA表达。采用结扎左前降支方法制备32只C57B/L小鼠心肌梗死模型,随机分成假手术组、模型组、常氧ADSCs移植组和低氧ADSCs移植组,两移植组在小鼠心肌梗死区边缘分别注射常氧细胞或低氧预处理细胞,于注射后4周采用免疫荧光法检测小鼠心肌细胞凋亡及血管再生情况。结果低氧组和常氧组细胞符合ADSCs特性,低氧组ADSCs Ki67表达阳性率高于常氧组(P<0.05),低氧组ADSCs PLGF、HGF、VEGF mRNA相对表达量均高于常氧组(P均<0.05)。细胞移植后4周,与模型组相比,ADSCs移植组小鼠心肌梗死区边缘caspase-3阳性凋亡细胞数降低,vw F阳性血管内皮细胞数升高,以低氧ADSCs移植组改变最明显(P均<0.05)。结论低氧预处理ADSCs移植能下调小鼠心肌梗死周边细胞凋亡反应,促进组织血管再生,其机制为低氧预处理能提升ADSCs的分泌功能。

Objective To investigate the effects of hypoxia-preconditioned adipose mesenchymal stem cells transplantation on the apoptosis and angiogenesis in a mouse model of myocardial infarction and to evaluate the underlying mechanism.Methods Primary cultured and identified mouse ADSCs at passage 3 were randomly divided into the normoxia group and hypoxia group,which were continued cultured in 21%and 2%O 2 for 6 days,respectively.The expression of specific mark proteins and Ki67 of the two group were investigated.The mRNA expression of vascular endothelial growth factor(VEGF),hepatocyte growth factor(HGF)and placental growth factor(PLGF)in the normoxia ADSCs and hypoxia ADSCs were evaluated by real-time PCR.Totally 32 mice were randomly divided into the sham group,control group,normoxia group and hypoxia group,and myocardial infarction models were established by ligating the left anterior descending branch of coronary artery.Immediately after injury,the mice in the normoxia group and hypoxia group were transplanted with normoxia ADSCs and hypoxia-preconditioned ADSCs on the edge of the infarcted area,respectively.The control group was injected with PBS,and the sham group were untreated.Immunofluorescence assay was used to detect the apoptosis and angiogenesis at 4 weeks after myocardial infarction.Results Immunocytochemistry results showed that cells in both normoxia group and hypoxia group had the characteristics of ADSCs.The positive rate of cell proliferation marker Ki67 in hypoxia-preconditioned ADSCs was significantly higher than that of normoxia ADSCs(P<0.05),the mRNA expression of PLGF,HGF and VEGF in the hypoxia group were significantly higher than that of the normoxia group(P<0.05).At 4 weeks after the cell transplantation,compared with the control group,there were significantly more vwF positive blood vessels and decreased caspase-3 positive apoptosis cells on the edge of infarct area in the ADSCs group.The most significant change was found in the hypoxia group(P<0.05).Conclusion Hypoxia-preconditioned ADSCs transplantation significantly decreases the apoptosis reaction and elevates the angiogenesis in the infarct border zone in mice with myocardial infarction by promoting the secretion of ADSCs.