【作者】 冯雪；

【导师】 罗国安；

【作者基本信息】 清华大学 ， 化学， 2005， 硕士

【摘要】 骨髓间充质干细胞(BMMSCs)是一种来源于骨髓具有自我更新和多分化潜能的细胞系,在体外可以扩增生长且不失去其特性。在特定的条件下,BMMSCs可以分化为成骨细胞、成软骨细胞、成肌细胞、上皮细胞和脂肪细胞等。因为BMMSCs易于获得,所以适合自体移植,成为细胞疗法和组织工程合适的种子细胞。因此,研究BMMSCs在特定条件下定向分化为心肌细胞对细胞移植治疗心肌梗死具有重要的理论及临床意义。本论文中我们分别从大鼠和小型猪骨髓中建立了体外分离纯化培养BMMSCs的基本模式和5-氮胞苷诱导其体外定向分化为心肌样细胞的模型,并且采用流式细胞技术、免疫组织化学染色和RT-PCR方法对诱导前后大鼠的细胞进行鉴定。结果表明:诱导前的细胞呈成纤维样,贴壁紧密,增殖迅速;在鉴定的7个细胞表面标志物中,CD29、CD44、CD105、CD166表达呈阳性,CD14、CD34、CD45表达呈阴性;细胞表达三个干细胞标志基因nestin、nucleostemin (NST)和Oct-4。BMMSCs重新贴壁72 h时添加10μmol/L 5-氮胞苷诱导剂,作用24 h后更换新鲜培养基继续培养,细胞形态逐渐变细长,增殖渐缓,4周后细胞表达心肌标志物cTnI、β-MHC和MLC-2v。这些结果很好的说明了我们从大鼠骨髓中分离纯化的细胞确实为间充质干细胞,并且经过5-氮胞苷诱导后可以分化为心肌样细胞。在以上研究基础上,采用益气活血中药含药血清干预小型猪BMMSCs体外增殖和分化,MTT法考察其增殖情况,蛋白组学方法考察对其分化的影响。结果显示:含药血清对BMMSCs的增殖在浓度小于10%时有一定的促进作用,否则则表现出抑制作用。双龙含药血清干预5-氮胞苷协同诱导BMMSCs体外定向分化,发现中药干预影响41个蛋白发生显著性变化,在中药干预时间进程中有18个蛋白发生显著性变化,变化的13个蛋白被成功鉴定。其中,PROX2、Hsp27、stathmin-1和BMP-7等蛋白在细胞增殖、分化过程中起重要的作用。结果说明益气活血中药含药血清对小型猪BMMSCs体外增殖与分化有一定的协同作用。更多还原

【Abstract】 Bone marrow mesenchymal stem cells (BMMSCs),which can be isolatedfrom bone marrow and expanded in vitro without any apparent modification inphenotype, are a group of cells with the capability of self-renewal and potentialof multilineage differentiation. Under certain conditions, BMMSCs candifferentiate into the osteogenic, chondrogenic, myogenic, endothelial andadipogenic lineages. As BMMSCs could be easily obtained, they are verysuitable for self-transplantation and have tremendous potential for cell therapyand tissue engineering. Accordingly, the investigation of the differentiation ofBMMSCs into cardiomyocytes is of great theoretical and clinical significationfor treating myocardial infarction by cell transplantation.In this study, we established the models of isolating and purifyingmesenchymal stem cells from marrow and inducing rat-BMMSCs todifferentiate into cardiomyocytes by 5-azacytidine in vitro, and identified thecells both before and after treated with 5-azacytidine using the ways of flowcytometry analysis, immunohistochemical method and RT-PCR. The resultsshowed that the original cells, which were a fibroblast-like morphology,attached to the culture dishes tightly, and proliferated quickly in the culturemedium, uniformly expressed the cell surface markers CD29, CD44, CD105 andCD166 but not CD14, CD34 and CD45, and also expressed the genes of stem cellmarkers nestin, nucleostemin (NST) and Oct-4. When cultured BMMSCs weretreated with 10 μmol/L 5-azacytidine for 24 hours on day 3 of a 4-week culture, thecells were lengthened in configuration and slowered in proliferation gradually, andexpressed the cardiac muscle cell markers cardiac-specific troponin I, β-myosinheavy chain and myosin light chain-2v after 4 weeks. All of above, we demonstratedthat plastic adherent cultures elaborated from rat bone marrow were mesenchymalstem cells, which could differentiate into cardiomyocytes by 5-azacytidinetreatment in vitro.On the basis of primary study, we investigated the effect of medicated sera ofTraditional Chinese Medicine (TCM) on the proliferation and differentiation ofswine-BMMSCs by MTT assay and proteomic approach, respectively. The resultsshowed that when the concentration of medicated sera was below 10% theproliferation of BMMSCs could be improved. However, when the concentration wasabove 10%, they could be suppressed. By studying the differentiation of BMMSCsinduced by 5-Azacytidine and intervened by Shuanglong medicated sera using theproteomic approach, we found the expressions of 41 proteins were remarkably alteredcompared with the control, and 18 proteins were remarkably altered during the courseof intervention. Of the 13 proteins which were subsequently identified, PROX2,Hsp27, stathmin-1 and BMP-7 may be involved in the cell proliferation anddifferentiation. Our study indicated that under certain condition, medicated sera ofTCM could promote the proliferation and 5-azacytidine-induced differentiation ofBMMSCs in vitro.更多还原