【作者】 许文荣；

【导师】 张锡然；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2004， 博士

【摘要】 骨髓间质干细胞（mesenchymal stem cells，MSCs）是一类成体干细胞，主要分布在骨髓基质中，属于非造血组织的间质干细胞，具有多向分化潜能。近年骨髓MSCs的多向分化潜能倍受关注。虽然在骨髓有核细胞中MSCs含量较少，但它的易贴壁性，分离纯化简单，扩增迅速，可长期传代，而且在不同的条件下能分化为多种组织，引起科学家的极大兴趣，尤其是骨髓MSCs定向分化为心肌、神经、骨细胞等，为细胞工程治疗心肌梗死、肌组织损伤、神经细胞和组织损伤、骨和软骨组织损伤提供干细胞的来源。 实验研究开展了成人、胚胎、猪骨髓MSCs的分离纯化及体外培养，成人骨髓MSCs在原代培养3d后，可见单个或少量成集落生长的贴壁细胞，形态大多呈短梭形或针尖状，10-15d后，细胞集落不断扩大并形成融合单层，大多呈长梭形或多角形，经传代后呈均匀分布生长，成人和胚胎骨髓MSCs体外扩增10代的细胞数约分别为（1～5）×10⁹和（5～10）×10¹²三种骨髓MSCs的基本形态相似，流式分析细胞表面标志，三种骨髓MSCs均呈CD13，CD29、CD44和CD71阳性，CD3、CD14、CD33、CD44、CD38和HLA—DR等为阴性。DNA含量和生长曲线显示，猪骨髓MSCs较成人和胚胎MSCs具有更强的增殖能力。成人、胚胎和猪MSCs体外可传代分别为10代、15代和40代以上。遗传学分析表明，在不同的传代时期三者的染色体核型没有改变。组织化学显示，三种骨髓MSCs呈POX、SB染色阴性，α-NAE、PAS呈强阳性，ALP绝大多数为阴性，只有1％左右为阳性。结果说明了体外骨髓MSCs基本生物学特性与体内一致。 首次采用5-氮胞苷（5-aza）等诱导成人第2代骨髓MSCs，培养2周后，细胞形状变细长并可融合产生肌管，电镜观察发现在诱导后细胞浆中有大量的肌丝样结构，免疫组织化学显示诱导后细胞呈α-肌动蛋白、β-肌球蛋白重链和结蛋白强阳性，约30％的细胞出现心肌细胞样分化。RT—PCR和实时荧光定量PCR结果提示在诱导后细胞中α-肌动蛋白、β-肌球蛋白重链、结蛋白和心肌肌钙蛋白T博士学位论文骨饭问质干细胞诱导分化及致瘤特性研究mRNA表达明显增加，而Q一肌动蛋白、结蛋白PCR扩增产物经TA克隆ONA测序证明其产物的准确性。通过激光共聚焦显微镜，可观察到诱导后细胞表现出节律性钙波动，在KCI的刺激下呈急剧上升趋势，随后出现下降。因此，可以认为成人骨髓MSCS在5一aza等的诱导下可以分化为心肌细胞。 采用p一琉基乙醇等诱导胚胎骨髓MSCS，诱导后细胞呈神经细胞的形态特点。免疫组织化学显示:胶质纤维酸性蛋白诱导后细胞呈神经丝蛋白（NF20O）、神经元烯醇化酶（NSE）、（GFAP）强阳性，并用述三种神经细胞标志的mRNA。结果表明用下分化为神经细胞。盯一PCR证明诱导后细胞明显地表达上胚胎骨髓MSCS能在p一琉基乙醇等作 采用抗坏血酸、p一磷酸甘油、地塞米松及bFGF诱导猪骨髓MSCs，结果表明，诱导后细胞形态由长梭形向多边形转变，ALP染色阳性，VonKoSSa染色阳性，呈典型的成骨细胞特点。初步证明猪骨髓MSCs能分化为成骨细胞。 采用电穿孔法将带有GFP的质粒（pGFP一NI）转染大鼠骨髓MSCS，结果显示电容在1050 pF电压260V时效果较好，转染率为50⁶0%，经过G418筛选，有限稀释法筛选阳性克隆，体外克隆后能稳定表达到10代，10代后荧光明显的减弱。这些结果说明GFP基因能在MSCs中进行有效的复制、转录和翻译，并可持续、稳定、高效表达。 采用淋巴细胞增殖检测法，发现体外胚胎MSCS能有效地抑制外周血淋巴细胞增殖反应，说明MSCs具有下调免疫的功能。胚胎骨髓MSCS能够和细胞因子（IL一3、SCF、FL、TPO）共同促进脐血CD34‘细胞的增殖，同时能够维持CD34‘细胞的数量，说明MSCS对扩增和维持CD34十细胞数量具有重要作用。 在诱导胚胎骨髓MSCs向树突状细胞的研究中，发现MSCS在IL一4和GM一CSF诱导下，细胞发生了突变，突变细胞变圆，失去了贴壁特性，但具有很强的增殖能力，细胞表面低表达CD13、CD29、CD44和CD91，遗传学分析显示染色体数量和质量的改变，主要表现为染色体着丝粒断裂。DNA含量分析显示出超二倍体细胞，与肿瘤细胞相似。我们克隆了这株突变细胞，并取名为F6，迄今F6细胞已培养17个月62代，细胞生长情况良好。SC工D裸鼠致瘤模型结果表明，F6细胞在皮下注射后9天出现肿瘤，随着时间延长肿瘤体积增大，同时3/8个SCID鼠发现有腹腔肿瘤细胞的转移。组织病理学检查发现，F6细胞为低分化肿瘤细胞，博士学位论文骨位间质干细胞诱导分化及致瘤特性研究并能广泛浸润周围的正常组织如真皮、肌肉组织、神经组织、脂肪组织及淋巴组织，对照组没有发现致瘤特征。F6细胞和人类肿瘤细胞株同样能表达nuCleostemin·Bmi一1·CATNNBI和Noteh一1基因，而且发现F6转移瘤组织高表达nueleostemin。 总之，本研究已分离纯化了成人、胚胎、猪骨髓MSCS，并研究了三种骨髓MSCS在体外生长、传代、细胞形态等生物学特性;并成功地将骨髓 MSCS体外诱导成心肌细胞、神经细胞和骨细胞;证明了MSCs具有免疫抑制和支持造血的作用，尤其是MSCS有效地体外扩增和维持CD34+细胞数量;发现了MSCS在IL一4和更多还原

【Abstract】 Mesenchymal stem cells (MSCs), a population of adult stem cells, are mainly located in bone marrow stroma. They are nonhematopoietic stem cells with pluripotency. Although the percentage of MSCs in adult bone marrow cells is very small, the multipotential of these cells, their characteristics of easy isolation and culture, as well as high ex vivo expansive potential make them an attractive therapeutic tool capable of playing a role in a wide range of clinical application. MSCs retain the ability to differentiate into various types of tissue cells and provide stem cells for the regeneration of various tissues including bone, cartilage, muscle and myocardium.MSCs were isolated and cultured from adult human, embryonic and porcine bone marrow and their basic biological characteristics were investigated. MSCs comprised a single phenotypic population and displayed a fibroblast-like morphology. After culturing for three days, MSCs began to adhere to culture flask and displayed spindle shape in culture. After ten days, it became confluent and fibroblast-like. After primary culture for ten passages, approximately (1~5) 109 and (5~10) 1012 cells were respectively obtained from adult human and embryonic bone marrow MSCs. The morphology of the three kinds of MSCs was similar.These expanded and attached MSCs were positive for CD13 CD29 CD44 CD71 and negative for GD14 CD33 CD34 CD38 CD45 HLA-DR, which were detected by Flow cytometer(FCM). Ten passages culture for adult human MSCs , 15 passages for embryonic MSCs and more than 40 passages for porcine MSCs were accomplished. The results of DNA contents and growth curve showed that porcine MSCs had stronger self-renewal capacity than that of adult human MSCs and embryonic MSCs. Karyotype of these MSCs did not change after long-time culture.These cells were positive for a -NAE, PAS and negative for POX, SB, ALP in the histochemical analysis. These results have demonstrated that MSCs have strong self-renewal capacity and can be cultured for long time.The adult human MSCs at passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. The differentiated MSCs were spindle shaped with irregular processes and formed myotube after two weeks’ treatment. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin and alpha-cardiac actin.Approximately 30% of all the remaining adherent cells had enlarged and assumed ball-like or stick-like morphologies and differentiated into a cardiomyocyte phenotype in vitro. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. RT-PCR, real-time quantitative PCR showed that mRNAs of beta-MHC, desmin, alpha-cardiac actin and cardiac troponin T were highly expressed in the myogenic cells. DNA sequencing results confirmed that the human cardiac troponin T and desmin genes amplified by PCR were identical to their respective reference sequences provided by GeneBank (X74819; NM 001927). The myogenic cells displayed both spontaneous rhythmic Ca2+ fluxes and KC1 induced Ca2+ fluxes when observed under confocal laser scanning microscope. The KCl-stimulated Ca2+ fluxes peaked rapidly and gradually declined thereafter. These results indicate that 5-azacytidine can induce human MSCs to differentiate into cells with characteristics commonly attributed to cardiomyocytes in vitro. Cardiomyocytes derived from bone marrow sources are potentially valuable for repairing injured myocardium.Differentiation of embryonic bone marrow MSCs into neural cells were induced with -mercaptoethanol. Neuron-specific enolase(NSE), neuronfilament 200(NF200), glial fibrilary acidic protein(GFAP) were positive in the differentiated cells detected by immuneohistochemistry. The mRNAs of NSE, NF200, GFAP in these cells were expressed higher than those of control group. The results showed that embryonic bone marrow MSCs treated with - mercaptoethanol in vitro could differentiate into neural cells.Porcine bone marrow MSCs were treated with ascorbic更多还原