目的:探讨整合素连接激酶(ILK)过表达对心脏c-Kit+细胞存活和增殖的影响,以及移植ILK过表达的心脏c-Kit+细胞改善心肌梗死(MI)大鼠心功能的作用.方法:从新生SD大鼠分离培养心脏c-Kit+细胞,构建含人ILK全长的腺病毒载体并感染心脏c-Kit+细胞.感染48 h后,应用细胞计数和CCK-8法测定细胞活力和增殖,并通过Western blot法检测c-Kit+细胞中增殖相关蛋白cyclin D1和增殖细胞核抗原(PCNA)的表达.40只8周龄SPF级大鼠经冠状动脉结扎建立MI模型,结扎15 min后于梗死区及梗死边缘区分3点通过心肌注射移植心脏c-Kit+细胞.采用随机数字表法将大鼠分为假手术组(sham组)、心梗+生理盐水注射组(MI组)、心梗+感染空载体的c-Kit+细胞注射组(Ad-null-c-Kit+ cell组)和心梗+感染携带ILK腺病毒载体的c-Kit+细胞注射组(ILK-c-Kit+ cell组),每组10只.术后2周,免疫组化法检测梗死区和梗死边缘区c-Kit的表达;术后4周,通过血流动力学检测心脏功能.结果:过表达ILK促进了心脏c-Kit+细胞的存活和增殖(P＜0.05).过表达ILK的心脏c-Kit+细胞中cyclin D1和PCNA的表达明显增加(P＜0.05).细胞移植2周后,ILK-c-Kit+ cell组心肌组织中c-Kit表达明显增加(P＜0.05);细胞移植4周后,ILK-c-Kit+ cell组大鼠心功能改善比Ad-null-c-Kit+ cell组更明显(P＜0.05).结论:过表达ILK通过提高cyclin D1和PCNA的表达,促进了心脏c-Kit+细胞的存活和增殖.ILK-c-Kit+细胞移植能更好地改善MI大鼠的心功能.%AIM:To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit + cells,and the role of ILK-overexpressing c-Kit + cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS:Cardiac c-Kit + cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit+ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA.The survival and proliferation of cardiac c-Kit + cells were detected by cell counting and CCK-8 assay at 48 h after infection,respectively.The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit+ cells were examined by Western blot.MI was induced by coronary artery ligation in 40 adult rats.After 15 min,ILK-c-Kit + cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone.All rats were randomly divided into 4 groups:sham group,MI plus saline injection group (MI group),MI plus null vector-infected cardiac c-Kit+ cell injection group (Ad-null-c-Kit+ cell group),and MI plus ILK-overexpressing cardiac c-Kit + cells injection group (ILK-c-Kit+ cell group),with 10 rats in each group.At 2 weeks after MI,the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay.At 4 weeks,left ventricular function was examined by hemodynamic measurement.RESULTS:The survival and proliferation of cardiac c-Kit + cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group.In MI rat model,the number of c-Kit + cells was increased by ILK-c-Kit + cell injection compared with Ad-null-c-Kit + cell group at 2 weeks after MI.Cardiac function was significantly improved in ILK-c-Kit + cell-transplanted rats.CONCLUSION:ILK overexpression improves survival and proliferation of cardiac c-Kit + cells by increasing the protein levels of cyclin D1 and PCNA.ILK-c-Kit + cell transplantation enhances the therapeutic efficiency of cardiac c-Kit + cells in the postMI hearts of rats.
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