摘要
目的 研究大鼠心包膜组织干细胞中分离、纯化血管内皮生长因子受体（KDR阳性）细胞的特性和心肌细胞的分化能力,为心包膜干细胞用于心脏细胞治疗提供理论依据.方法 取Wistar大鼠心包外脂肪,I型胶原酶消化法获取原代干细胞,培养并适时传代;然后分别用流式细胞仪和免疫组化方法检测分离细胞的免疫表象,并进一步用磁珠分离法分离出KDR阳性细胞,用5-氮杂胞苷诱导其向心肌细胞分化,分化后的细胞表型用免疫组化分析检测.结果 分离细胞成梭状,体外培养呈良好增长.FACS分析显示,这些细胞具有间充质干细胞的细胞特征,分别为CD29、CD44、CD90阳性,部分呈CD106阳性,CD34 和CD45阴性.免疫荧光染色显示,分离的干细胞为isl-1阴性,少量的c-kit、GATA-4阳性,约3%~5% 为KDR阳性细胞.磁珠分离法分离KDR阳性细胞,在药物诱导7d后,部分细胞聚集成细胞团,可见单个细胞开始自发性博动.免疫组化染色可以观测有心肌特异蛋白和结构蛋白的表达,并观察到明显的肌小节结构.结论 心包组织干细胞具有充间质干细胞的基本特征,并具有多样性的免疫表象,其中以KDR阳性细胞较多.纯化的KDR阳性细胞具有定向心肌细胞分化的潜能,提示其可以作为用于心脏细胞治疗的供体细胞来源之一.
Objective Analyzed the biological properties and eardiomyogenic differentiation of KDR＋ cells derived from the pericardial tissue in rats. Methods Cells were isolated by enzymatic digestion of pericardial tissue and their immunological phenotypes were analyzed by using FACS and immunohistological staining. After purified by magnetic activated cell sorting, a subpopulation of KDR positive cells （KDR＋） was pharmacologically induced towards oardiomyogenic differentiation. Results The pericardium-derived stem cells grew exponentially and exhibited a similar morphology to cardiosphere. FACS analysis revealed positive for CD29,CD44,CD90 and CDlO6,and negative for CD34 and CD45. Meanwhile, small fraction of isolated cell also showed KDR, c-kit and GATA-4 positive. The purified KDR＋ cells underwent cardiomyogenic differentiation after pharmacological induction, evidenced by single cell spontaneous contraction and expression of cardiac structural proteins （cTnT and a-MHC）. Conclusion The pericardium-derived adipose stem cells are heterogeneous cell population of mesenchymal stern cells, in which KDR＋ cells exhibited differentiational potential towards beating cardiomyocytes. Therefore KDR＋ cells may represent an attracting cellular candidate for cardiac cell therapy.
出处
《浙江医学》
CAS
2012年第2期91-94,共4页
Zhejiang Medical Journal
基金
浙江省科技计划项目（2010C33137）
