大鼠心包膜组织WT1阳性细胞的分离、培养和生物学特性(附视频)  

Pericardial tissue-derived WT1 positive cells: isolation, expansion and biological characteristics (video)

作　　者：王晓明[1] 许林海[1] 聂良明[1] 孟群[2] 

机构地区：[1]浙江省人民医院心胸外科,杭州310014 [2]浙江省人民医院心病理科,杭州310014

出　　处：《中华移植杂志（电子版）》2012年第2期30-34,共5页Chinese Journal of Transplantation(Electronic Edition)

基　　金：浙江省科技计划项目基金(2010C33137);浙江省科技厅公益技术研究社会发展项目(2012C23078)

摘　　要：目的了解大鼠心包膜组织中分离和培养干细胞的可行性、细胞特性和分化成心肌细胞的能力,为心包膜组织干细胞用于心脏疾病细胞治疗提供理论依据。方法取Wistar大鼠制备心肌梗死模型后取心包组织,胶原酶消化法分离心包膜组织干细胞,并进一步梯度离心纯化,培养并适时传代,测定其生长曲线;分别用流式细胞仪和免疫组织化学法检测分离细胞的免疫表型。用5-氮杂胞苷诱导心包膜组织干细胞向心肌细胞分化,分化后的细胞表型分别用免疫组织化学法和反转录PCR分析检测。结果分离细胞成梭状,体外培养呈良好增长,组织形态与心脏干细胞相似。免疫表型呈CD29、CD44、CD90阳性,部分呈CD106阳性,而CD34和CD45阴性;95%为WT1阳性细胞,并有少量的c-kit、心脏肌钙蛋白T、GATA-4、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,可以观测到明显的肌小节结构,并同时有心源性转录因子和结构蛋白在转录水平表达。结论心包膜组织干细胞具有间充质干细胞的基本特征,并具有多样免疫表型,其中以WT1阳性细胞为主;纯化的WT1阳性细胞具有定向分化心肌细胞的潜能,提示心包膜组织可以作为心脏疾病细胞治疗的供体来源之一。Objective To analyze the biological characteristics and cardiomyogenic differentiation of stem cells derived from the pericardial tissue in rats. Methods Pericardial tissue in Wistar rats was harvested after the establishment of myocardial infarction. Cells were isolated by enzymatic digestion of pericardial adipose tissue and their immunological phenotypes were analyzed by using FACS and immunohistological staining. After Ficoll gradient centrifugation, a subpopulation of WTI positive cells was pharmacologically induced towards cardiomyogenic differentiation. Results The pericardium-derived stem cells grew exponentially and exhibited a similar morphology to cardiosphere. FACS analysis revealed they were positive for CD29, CD44, CD90, and CD106, and negative for CD34 and CD45. Meanwhile, large fraction of isolated cells also showed WT1 positive staining （95%）, while only a limited proportion showed KDR, c-kit, cTnT, and GATA-4 positive staining. The purified WT1 positive cells underwent cardiomyogenic differentiation after pharmacological induction, evidenced by single cell spontaneous contraction and expression of transcriptional factors for cardiogenesis （GATA-4 and MEF2） and cardiac structural proteins （cTnT and α-actinin）. Conclusion The pericardium-derived stem cells are heterogeneous cell population of mesenchymal stem ceils, in which WT1 positive ceils exhibited differential potentials towards beating cardiomyocytes. Therefore WT1 positive cells may represent an attractive cellular candidate for cardiac cell therapy.

关 键 词：心包 干细胞 WT1 心肌细胞 细胞分化 视频 

分 类 号：R541[医药卫生—心血管疾病]