【作者】 杨博；

【导师】 刘苏；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的心肌细胞丢失、瘢痕形成及心室重构是造成心脏功能恶化的主要因素,也是最终导致慢性充血性心力衰竭的主要病理基础。细胞移植治疗是治疗心脏疾病的一种新策略。骨髓间充质干细胞(BMSCs)目前被认为是细胞移植治疗最理想的种子细胞。研究BMSCs体外定向分化为心肌样细胞对细胞移植治疗心脏疾病具有重要的理论和临床意义。本课题旨在通过血管紧张素Ⅱ化学诱导等方法体外诱导BMSCs定向分化,检测BMSCs是否可分化为心肌样细胞,观察血管紧张素Ⅱ对BMSCs生长增殖的影响,对BMSCs分化的可能机制作一探讨。本研究以人骨髓间充质干细胞为研究对象,实验分为两部分,第一、体外分离、纯化、传代人骨髓间充质干细胞(HBMMSc)。第二、血管紧张素Ⅱ体外对HBMMSc向心肌样细胞的诱导分化的研究。方法1 HBMMSc的分离,培养和传代及鉴定标本来源于正常人的骨髓。采用淋巴细胞分离液行密度梯度离心法分离骨髓单个核细胞,再利用贴壁筛选法纯化骨髓间充质干细胞后,进行原代和传代培养,并对其进行细胞生长和形态学的观察、表面抗原的鉴定。2 HBMMScs在血管紧张素Ⅱ的诱导下向心肌细胞的培养分化及心肌细胞的鉴定。收集第8代hBMMSCs,分4组为实验组3组和空白对照组。用0.1μmol/L AngⅡ诱导孵育hBMMSCs 24h。通过倒置显微镜形态学观察、半定量逆转录-聚合酶链反应(RT-PCR)法鉴定诱导细胞是否具备心肌样细胞的形态、结构特点及是否表达心肌特异性蛋白cTnI,GATA-4,Nkx2. 5。结果1 hBMMScs原代培养接种24h贴壁完成,2-3天细胞呈梭形,第9天形成多个克隆,第14-16天细胞融合成单层,梭形突起变长,排列有明显方向性,细胞排列成漩涡状。传代细胞24h内完全贴壁,伸展成梭形,开始迅速增殖,7天即铺满培养瓶底,传代细胞保持原代细胞的形态特征。随代数增加,细胞得到纯化,梭形细胞达95%以上。连续传代至P8,细胞由梭形变为平坦、宽大,分裂相减少,细胞质疏松,可见空泡。传至P10,部分细胞变成圆形,折光增强,脱壁死亡。随着传代次数的增加,克隆形成率逐渐下降,10代后无明显克隆出现。流式细胞测定分离提纯后的hBMMScs表达CD29和CD44,不表达CD34和CD45。2 AngⅡ诱导的细胞第8-l0d即呈棒状,第18-21d细胞为长杆状,走向便趋一致。电泳检测证实AngⅡ诱导的细胞第2、3、4、5周已经表达cTnI,NkKX2.5,GATA-4,并且随时间推移,蛋白表达增强。空白对照组hBMMSCs无形态变化,不表达心肌特异蛋白。结论1利用淋巴细胞液进行密度梯度离心法和贴壁筛选法,可分离纯化hBMMSCs,细胞的均一性高,是一种简单,经济有效的分离提纯hBMMSCs方法。并且扩增细胞数量足够,遗传背景稳定,可满足组织工成要求。体外分离、培养hBMMSCs具有很好的成功率及成活率。2 AngⅡ在体外可诱导第8代hBMMSCs分化为心肌样细胞。更多还原

【Abstract】 ObjectivesCardiomyocytes loss, myocardial infarction emergen and ventricular remodeling are not only the leading causes of pump disfunction, but also primary pathomechanism of congestive heart failure. Cellular transplantation is being explored as a newly developed strategy for treating patients with end-stage heart disease, and bone marrow mesenchymal stem cells (BMSCs) have been proposed as a type of ideal candidate. Research on BMSCs directional cardiomyogenic differentiation ex vivo can offer great theoretical and the clinical significance for cell therapy used in heart diseases.For this reason the present study investigated angiotensinⅡinduced cardio- myogenic differentiation of HBMMSCs in vitro and determined the alterations of growth properties in HBMMSCs produced by angiotensinⅡ. We also evaluate the underlying mechanisms of BMSCs commitment cardiomyo- genic differentiation.The experiment study the HBMMScs.It has two parts.To isolate and proliferente human bone marrow-deserve mesen- chymal stem cell.To study inducing function of angiotensinⅡon human bone marrow-derived mesenchymal stem cell.MethodsPartⅠIsolation, culturing and identifying of HBMMSCs the samples came from human bone marrow. The methods of density gradient centrifugation and adherent filtration were used to isolate and purify HBMMSCs. HBMMSCs were cultivated. The cellular growth status and morphology were observed, and surface antigens of HBMSCs were tested.To aspirates the marrow and extract the hBMMSCs accordingthe principle of density gradient centrifugation,then culture and amplify the hBMMSCs.Part II: To collect passages 8cells,then classify four groups,Three groups is induce by 0.1μmol/L Ang II.One group is placebo. To identify the cells feature of morphous and construction and special protein by inverted microscope, reverse transcription-polymerase chain reaction.Results1 After 24 hours in primary culture, the cells adhered to the plastic surface. After 2-3 days, the cells were shuttle-shape. The cells began to form a few clones at day 9. The cells fused into monolayer and the shuttle-shape cells became long, obvious directivity and eddy-shape. After 24 hours, the cells of passage adhered to the plastic surface, extended to become shuttle-shape and began to proliferate rapidly. After 7 days, the cells overspread the culture bottle bottom, and kept the same figure characteristics of primary culture. With passage’s number increasing, the cells were purified and the shuttle- shape cells were over 95%. When the cells came to the eighth passage, shuttle-shape of them changed to plainness and bounty, cytokinesis decreased, and cytoplasm was loosen and there were vacuoles in cytoplasm. When came to the tenth passage, the part of the cells became round, refraction of them increased and some of them died. With passage’s number increasing, the clone formation ratio decreased gradually. After the ten passages, the new clones weren’t found. HBMMSCs surface antigen profiles obtained by flow cytometry were positive for CD29, CD44 and negative for CD34 and CD45.2 AngⅡgroup, eight to ten days after treatment ,soon cell gradually increase in size and formed a ball-like or stick-like appearance. The induced cells of positive experimental group were stained positively for GATA-4 Nkx2-5and cTnI. RT-PCR (Reverse Transcrip-Polymerase Chain Reactin) also showed positive experimental group cells expressed GATA-4 Nkx2-5 and cTnI.Conclusions1 The methods of density gradient centrifugation and adherent flitration were simple,economic and efficient to isolate and purify HBMMSCs.After isolation,purifying and proliferate,the number of HBMMSCs were enough and hereditary back- ground of were steady to suit for tissures engineering. isolated and cultured in vitro successfully. 2 HBMMSCs can be induced into cardio myocyte like cells by AngⅡ更多还原