【作者】 伍金红；

【导师】 谭玉珍；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2011， 硕士

【摘要】 目的建立骨髓胚胎样干细胞(ELSCs)分离与扩增方法,明确ELSCs的生物学特性及分化潜能,探讨ELSCs移植对心肌梗塞后心肌修复和心功能改善中的作用。方法用阶段特异性胚胎抗原1 (SSEA-1)标记骨髓单形核细胞,流式细胞仪分选ELSCs。透射电镜和扫描电镜观察ELSCs的超微结构特征,免疫细胞化学染色和RT-PCR检测胚胎干细胞(ESCs)转录因子在ELSCs的表达,将ELSCs在小鼠胚胎成纤维细胞饲养层上扩增,进行三胚层细胞分化能力检测,并用细胞因子诱导ELSCs向心肌和内皮细胞分化,检测分化细胞特异蛋白和mRNA的表达。将ELSCs移植到裸鼠皮下观察其致瘤性。建立大鼠心肌梗死模型,于梗死区边缘移植ELSCs,超声心动图检测心功能变化,图像软件分析瘢痕面积和胶原纤维含量的变化,Y染色体原位杂交和免疫组织化学染色观察移植细胞的存活及向心肌细胞和内皮细胞的分化,综合评价ELSCs移植后治疗心肌梗塞的效果,结果ELSCs约占骨髓单形核细胞的0.015%。电镜下可见ELSCs细胞核大,以疏松的常染色质为主,胞质少,核质比高。ELSCs表达Oct4、Nanog、Sox2和Rex1等ESCs转录因子。在普通培养条件下ELSCs增殖不明显,而在饲养层上培养时细胞增殖显著,呈簇状生长。在软琼脂中培养2w左右可见拟胚体样小体形成,细胞自然分化后4 w可检测到Nestin、Brachyury和E-cadherin等三胚层标志物的表达。用细胞因子诱导后4 w,分化的细胞表达心肌细胞特异蛋白cTnT和Cx43以及内皮细胞特异蛋白VWF和CD31。将ELSCs移植入裸鼠皮下后4 w,未观察到肿瘤组织或畸胎瘤形成。在体动物实验研究结果显示,移植预诱导的ELSCs,心功能得到明显改善,瘢痕面积显著减小,胶原纤维含量下降。Y染色体原位杂交和免疫组织化学染色显示Y染色体阳性细胞表达cTnT和Cx43,可见Cx43定位于Y染色体阳性细胞和宿主心肌细胞连接处。Y染色体阳性细胞同时表达CD31,且在预诱导的ELSC组中可见移植的细胞位于新生血管壁上。结论大鼠骨髓中存在ELSCs,这类细胞具有类似ESCs的生物学特特征,表达ESCs转录因子、在饲养层上有很高的增殖能力,在软琼脂中能够形成拟胚体样小体并能分化形成三胚层来源的细胞。在体外诱导条件下,ELSCs能分化形成心肌细胞和内皮细胞。移植入心肌梗塞动物模型后,预诱导的ELSCs能更有效地分化形成心肌细胞和内皮细胞,并可直接参与血管新生,ELSCs移植可通过促进心肌分化和血管新生改善心功能。因此,ELSCs有望成为干细胞研究和再生医学理想的种子细胞。更多还原

【Abstract】 Objective:Myocardial infarction (MI) is a major health problem and the leading cause of death and disability in industrialized and developing nations. Stem cell therapy is a promising therapeutic strategy for treating MI. However, it is important to select optimal stem cells and explore effectiveness and mechanisms after transplantation. Methods:Bone marrow mononuclear cells were labeled with SSEA-1, the positive cells were sorted by flow cytometry. The biological characteristics of ELSCs were studied in many aspects, the ultrastructural characteristics were observed under transmission electron microscope (TEM) and scanning electron microscope (SEM), the expreesion of transcriptional factors of embryonic stem cells (ESCs) in ELSCs were detected by immunocytochemistry and RT-PCR, prliferative ability was studied on feeder of mouse embryonic fibroblasts (MEF), formation of embryoid-like bodies were observed in agarose and the expression of trilineage markers was dectected in the cells of embryoid-like bodies, the possible formation of tumor and teratoma of ELSCs was investigated by injection into subcutaneous of severe combined immunodeficiency mice. Differentiation of ELSCs toward cardiomyocytes and endothelial cells was induced by cytokines. The expression of specific protein and mRNA was detected in induced cells. On the basis of in vitro experiments, differentiation of ELSCs in vivo and therapeutic effects was explored. MI female rat models were transplantated by PBS, MMSCs, ELSCs and induced-ELSCs. The cardiac function was examed by echocardiography after transplantation. Scar area was evaluated by Masson’s trichrome staining. The survival of the transplanted cells was detected by Y chromosome in situ hybridization. The differentiation of cardiomyocytes and endothelial cells in vivo was evaluated by the colocalization of the Y chromosome and cTnT, Cx43 and CD31. Results:ELSCs had typical features for ESCs, the nucleus was large, the ratio of nucleus to cytoplasm was very high under TEM, immunocytochemistry and RT-PCR analysis demonstrated that ELSCs expressed transcription factors Oct4, Nanog and Sox2, the cells proliferated significantly of feeder of MEF, the cells formed embryoid-like bodies after incubation in agarose with LIF and bFGF and expressed Nestin, Brachyury and E-cadherin. However, anatomical observation showed there was neither tumor nor teratoma in the body for 4 weeks after transplantation of ELSCs for SCID mice. After induction with cytokines for 4 weeks, ELSCs expressed cTnT and Cx43 for differentiation toward cardiomyocytes and expressed CD31 and vWF for differentiation toward endothelial cells. After transplantation for MI female rat models, cardiac function was improved and scar area was reduced significantly in the induced-ELSC group as compared with control, non-induced ELSC and MMSC groups. In induced-ELSC group the differentiated cells were parallel alignment and connected with host cardiomyocytes. Y chromosome fluorescence in situ hybridization demonstrated that the positive cells were colocalizated with cTnT and Cx43. Cx43 was located between Y chromosome positive cells and recipient cardiomyocytes. This structural basis was the premise for synchronized contraction of transplanted cells with host cells. Colocalization of the Y chromosome and CD31 revealed that transplanted stem cells also underwent endothelial cells differentiation in the infarcted and peri-infarcted regions. And ELSCs were directly participated in angiogenesis in infarcted and peri-infarcted regions. Conclusions:This is the first time to isolate ELSCs from adult rats. ELSCs have similar biological characteristics with ESCs, ELSCs express transcriptional factors Oct4, Nanog and Sox2, proliferate significantly on feed of MEF, form embryoid-like bodies in agarose and the cells in the embryoid-like bodies can differentiate into cells of trilineage. ELSCs can differentiate into cardiomyocytes and endothelial cells under induction with cytokines. Cardiac function is significantly improved, scar area is reduced and ventricular remodeling is effective controlled by transplantation of induced ELSCs. The transplanted cells differentiate into cardiomyocytes and endothelial cells, and the cells are directly participated in angiogenesis. These biological characteristics enable ELSCs to be an optimal source of seed cells for stem cell research and regenerative medicine, and this study provide a theoretical basis for MI by ELSC transplantation.更多还原