Apelin促进脂肪间充质干细胞增殖、分化修复小鼠下肢缺血损伤的实验研究

目的:探讨apelin对于脂肪间充质干细胞(AD-MSCs)修复小鼠后肢缺血损伤的作用及机制。方法: 从β-actin-luc转基因小鼠中分离出表达荧光素酶的AD-MSCs，将20只近交系无报告基因的小鼠随机分为两组，即实验组:后肢注射AD-MSCs+apelin(n=10)和对照组:后肢注射AD-MSCs(n=10)。所有小鼠均结扎股动脉建立后肢缺血模型，股四头肌内注射AD-MSCs(1×106)或AD-MSCs+apelin，激光多普勒成像观察下肢血液灌注，生物发光成像检测细胞的活性。用体外培养的β-actin-luc转基因小鼠AD-MSCs，建立缺氧（6 h）模型。实验分为4组，即对照组、缺氧组、缺氧＋apelin干预组及缺氧＋apelin+LY294002组。用免疫印迹法检测细胞内激酶磷酸化Akt 的表达。结果: 在移植后1周内生物发光成像显示，AD-MSCs移植后存在急性死亡，加用apelin组中细胞的存活时间明显延长。离体实验显示，在缺氧环境中apelin干预AD-MSCs后，细胞pAkt的表达明显高于对照组(P<0.05)，加入Akt阻断剂LY294002后，细胞磷酸化的水平降低(P<0.05)。结论: apelin对AD-MSCs的后肢缺血治疗具有保护作用，可能在缺血性疾病的治疗中成为重要的干预靶点。

In vivo visualization of apelin in survival and function of adipose-derived mesenchymal stem cells in hind limb of ischemic mice

AIM:To evaluate the contribution of apelin, a novel peptide with significant cardioactive properties and the therapeutic efficacy of mesenchymal stem cells in hind limb ischemia mice. METHODS: Adipose-derived mesenchymal stem cells (AD-MSCs) expressing firefly luciferase (Fluc) were isolated from β-actin-luc mice and characterized by flow cytometry and bioluminescence imaging (BLI). Male FVB mice underwent femoral artery ligation and received AD-MSCs (1×106) or AD-MSCs with apelin intraquadriceps femoris muscle injection. Cell survival was imaged by BLI and expressions of Akt and pAkt after cellular therapy were analyzed by Western blot. RESULTS: In vivo BLI revealed acute donor cell death of AD-MSCs within 1 week after transplantation. In contrast, signals of injected cells were still present after 1 week in AD-MSCs in apelin group (P<0.05). In vitro apelin treatment of AD-MSCs exposed to hypoxia increased cell proliferation and considerable increases in phosphorylation of Akt were found in AD-MSCs pretreated with apelin. CONCLUSION: Apelin has beneficial effects on the therapeutic efficacy and survival maintenance of AD-MSCs in hind limb ischemia and may constitute an important target therapy in ischemic disease.