【作者】 刘维新；

【导师】 程邦昌； 陈锡昌；

【作者基本信息】 武汉大学 ， 外科学， 2004， 博士

【摘要】 细胞移植作为一项极具开创性意义的技术和方法，为临床治疗心肌梗死带来了新的希望。近年来，有学者发现，骨髓基质细胞(marrow stromal cells，MSCs)具有可通过常规的骨髓穿刺而简单获取、强大的自我更新能力以及用胞嘧啶类化学物质5-氮胞苷诱导后或在心肌环境中可向心肌细胞分化的潜能。因此，MSCs被认为是细胞移植治疗心肌梗死最有前途的自体细胞来源之一。然而，MSCs向心肌细胞分化的潜能及具体机制尚不清楚；值得注意的是，目前已有越来越多的学者对成体干细胞是否具有广泛的分化潜能提出质疑。为深入研究和了解MSCs向心肌细胞分化的潜能及其临床应用价值，探索临床以细胞移植和基因工程治疗心肌梗死的新思路和新方法，本课题从下述几个方面进行研究： 1．Wistar大鼠MSCs体外培养的形态学和增殖动力学研究 为研究和观察MSCs体外培养时的形态学变化和增殖动力学特征，本部分实验采用Wistar大鼠作为实验动物，用离心法分离全骨髓，经原代培养24h后，用Hank’s平衡盐溶液(Hank’s balanced salt solution，HBSS)轻轻冲洗3次以去掉非贴壁的造血干细胞，可见贴壁的MSCs呈尖梭形散在分布。第3～4d后，MSCs呈集落样迅速增殖；6d后，不同大小的细胞集落明显增多(8～12个／60-mm皿)；第12d时，集落间彼此接触以致每个集落中央的细胞长至数层时，细胞几乎停止生长，但此时整个皿底并未被细胞铺满。传代的MSCs体积增大，形态上也表现出异样性，基本上可以分成两种类型：小的尖梭形或三角形及大的扁平形。前者随着不断的传代具有转变成后者的倾向；而且一旦转变成后者，增殖能力明显下降。第1代末时约15％～18％的细胞呈大的扁平型，而至第4代末时此类细胞超过57％。 当细胞以2845 cells/cm~2的密度接种时，在30d的传代培养过程中，细胞倍增数可达21.95。随着传代的进行，细胞增殖能力明显降低。原代培养的MSCs前3～4d保持在静止期，而后迅速增殖，至第10d时平均细胞数可达(15.37±0.10)×10~4个。10d后若不进行传代，细胞几乎停止增殖。第1代和第2代细胞的生长曲线与原代相似，静止期明显缩短，细胞数量在短期内增加更为明显(第1代约为4一7d:第2代约为4一6d):第3代的细胞增殖速度明显减慢，至第4代时细胞基本上停止增殖。 本部分实验结果表明，体外培养的MSCs其增殖能力有限，易于趋向衰老;其心肌细胞分化潜能、作为细胞移植的靶细胞及以其为载体的基因治疗的临床应用价值尚待进一步研究。2.体外胞啥健类化学物质5一氮胞昔诱导MSCs向心肌细胞分化的 研究 为验证体外胞喀睫类化学物质5一氮胞昔能否诱导MSCs向心肌细胞分化，本实验用不同浓度(3、5、10件mol几)的5一氮胞昔以不同的方式(一次处理与反复处理)诱导MSCS，通过对形态学的观察并采用免疫组织化学、从尾stem blot及心室肌特异性肌球蛋白轻链(ventrieular myosin light ehainZ，MLCZv)启动子控制下的蓝绿色荧光蛋白(enhaneed eyan variant ofAe口uorea巧etoria green fluoreseent Protein，ECFp)报道基因表达技术进行检测，以验证和分析5一氮胞昔诱导MSCs向心肌细胞分化的可行性。 实验结果显示，5一氮胞昔处理24h后，部分MSCs出现状态不佳甚至死亡的现象，而且随着药物浓度的提高，这种现象更加明显;各5一氮胞昔处理组的MSCs在整个3Od的观察过程中，既没有见到自发搏动的细胞，也没有见到肌管形成。当MSCs生长至融合时，可见“细条样”和/或“肌管样”结构形成，但这种结构即使在非5一氮胞营处理组，当细胞生长至融合时，同样可以出现。 免疫荧光检测表明，作为阳性对照的心肌细胞在荧光显微镜下可见明显的心肌特异性肌节肌球蛋白重链(myosin heavy ehain，MHC)及肌钙蛋白(troponinl，Tnl)的表达，在高倍镜下其横纹结构明显可见;然而这两种心肌特异性蛋白在所有5一氮胞普处理组的MSCs均未检测到表达，而且那些“细条样”和/或“肌管样”结构也不表达这两种蛋白。W七stem Blot检测结果提示，各5一氮胞普处理组MSCs经SDS一队GE后的免疫印迹结果没有发现心肌特异性MHC、Tnl蛋白阳性表达，而在作为阳性对照的心肌细胞蛋白的位置经免疫印迹显色后可见二者明显的条带。ECFP报道基因检测结果提示，经转染pMLCZv尼CFP的正常心肌细胞，48h后在荧光显微镜下(激发波长400士20nln)观察到了明亮的蓝绿色荧光;但在30d内，各5一氮胞昔处理组均未见呈蓝绿色荧光的MSCs出现。 本部分实验结果表明，先前学者们所报道的5一氮胞昔能够诱导MSCs向心肌细胞分化的作用并不具有可靠的重复性。尽管本实验不能排除长期反复用5一氮胞营处理可能导致极少数MSCs向心肌细胞分化，并通过筛选获得心肌生肌细胞株(eardiomyogenie eell line，CMG eell line)，但不宜简单地夸大5一氮胞昔对MSCs向心肌细胞分化的诱导作用。3.体外气心肌样”环境对MSCs向心肌细胞分化的诱导作用 为研究体外“心肌样”环境对MSCs向心肌细胞分化的诱导作用，本实验采用荧光染料如一二乙酞基一2一苯基叫噪(4，6一diamidino一2一phenyli耐ole，nAPI)预先标记MSCs，并将其与新生大鼠心肌细胞进行共更多还原

【Abstract】 Cell transplantation, as a revolutionary idea, has opened a new exciting perspective for the treatment of infarcted myocardium. Recently, bone marrow stromal cells (MSCs) have been considered as a promising candidate because of their some advantages such as easiness to obtain by a simple routine bone marrow aspiration, ability to self-renew and recently reported novel potential to differentiate into cardiomyogenic cells with the induction of 5-azacytidine or when exposed to a cardiac milieu. However, the precise understanding of the molecular control on this differentiation awaits some further experimental work, and the claim that adult stem cells can be converted into multiple unexpected cell types is also questioned by some experts. To give a further investigation on the potential of cardiomyogenic differentiation of MSCs and explore a new strategy for the therapy of myocardial infarction with cellular transplantation and gene engineering, the present study was performed in several parts as following:1. Morphology and Growth Properties of MSCs in vitro from Wistar RatsTo investigate the morphologic characteristics and proliferation kinetics of MSCs in culture ex vivo, the full marrow was isolated by centrifugation in vitro from wistar rats. After a primary culcure of 24 h, MSCs were seen to attach to culture dishes sparsely after discarding the nonadherent cells by the first medium change and by washing with Hank’s balanced salt solution (HBSS) three times. And the majority of the cells displayed a spindle-like shape. These cells began to proliferate at about day 3 or day 4, and gradually grew to form small colonies. By day 7, the number of cellular colonies with different size had obviously increased (about 8-12 colonies /60-mm dish). As growth of cells continued, colonies gradually expanded in size with the adjacent ones interconnected with each other. However, the cultures failed to reach confluency by day 12, although the cells in thecenter of the colonies had formed several overcrowded layers and nearly ceased proliferation at this time. Passaged MSCs were larger in size and more heterogeneous in morphology and in growth properties. Grossly, the MSCs in subcultures could be divided into two types: small spindle- or triangle-like, and broad flattened cells. The flattened cells seldom proliferated and it seemed that the spindle- and triangle-like MSCs gradually transformed into broad flattened cells with passages. At the end of the first passage, about 15-18% of cells were flattened, but at the end of passage 4, at least more than 57% broad flattened cells were observed.When seeded at a density of 2 845 cells/cm2 and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. For primary culture, the cells remained quiescent during the first 4 days of culture, and then quickly replicated until day 10 when the average cell number reached (15.37 + 0.10) X 104. The number of cells did not significantly increase during the following 4 days of culture (days 11 ~ 14). The patterns of the growth curves of MSCs in passages 1 and 2 were similar to those of primary cells. However, the cells showed a shortened quiescent period (days 1 ~ 3) before proliferation and the cell number kept increasing significantly for a shorter period (days 4 ~ 7 in PI and days 4 - 6 in P2). The increase of average cell numbers was considerably slowed in passage 3, and the cells almost ceased proliferation in passage 4.The present study proved that rat MSCs could not be extensively expanded in vitro and they were easy to become senescent with passaging; and the multidifferentiation potential, the application values for cellular transplantation as target cells and vectors of gene delivery shoud be questioned and investigated furtherly.2. The Investigation on the Effects of 5-Azacytine as a Cytosine Analogue on the Cardiomyogenic Differentiation of MSCsTo investigate whether a更多还原