【作者】 姜丽丽；

【导师】 刘天庆；

【作者基本信息】 大连理工大学 ， 化学工程， 2013， 博士

【摘要】 脂肪间充质干细胞(Adipose derived mesenchymal stem cells, AD-MSCs)和脐带间充质干细胞(Umbilical cord mesenchymal stem cells, UC-MSCs),由于来源广泛、不受争议,而成为治疗心脏疾病、修复心肌组织等细胞治疗的首选细胞。细胞治疗所需的时间很长,一般都在一个月以上,时间是影响细胞治疗效果的一个重要因素。此外,MSCs在移植部位保持高的细胞活性和活率对细胞治疗效果提高也尤为重要。然而,由于移植区域恶劣环境(如：局部缺血、氧化应激、炎症反应等)的作用以及一次性植入大量细胞产生细胞接触抑制而导致MSCs大量的凋亡或死亡,从而降低了细胞治疗效能。因此,寻找一种更能够耐受接触抑制的MSCs,应用于细胞治疗,从而提高细胞治疗的效果是必要的。另外,AD-MSCs和UC-MSCs能够向心肌细胞分化,并表达心肌细胞的特异性蛋白。然而,现有的化学诱导以及心肌细胞培养液诱导等方法,都很难使UC-MSCs或AD-MSCs分化成具有心肌细胞功能特性(细胞收缩和钙瞬变等)的细胞。因此,有必要寻找一种适宜的生物活性因子,使其与心肌诱导剂相结合,以促进UC-MSCs和AD-MSCs向心肌的功能性分化。本文首先研究了AD-MSCs和UC-MSCs在一个月连续培养过程中的生长增殖特性。通过CCK-8和血细胞计数板计数,分别考察了AD-MSCs与UC-MSCs的细胞活性及细胞数随时间的变化曲线；利用死活染色以及流式细胞仪技术,检测了两种来源的MSCs在一个月连续培养过程中的死活情况和细胞活率的变化情况。结果表明,在一个月连续培养过程中,AD-MSCs始终保持很高的细胞活性、细胞活率、以及增殖能力,细胞经历了2个平台期和3个对数增殖期,具有耐接触抑制的能力。而与AD-MSCs相比,UC-MSCs则没有始终保持很高的细胞活性,随着培养时间增加,尤其到培养后期时,其细胞活性明显下降,细胞活率也大幅降低,并且与同一时间点上AD-MSCs的细胞活率相比,具有显著性差异,细胞发生了接触抑制,基本不再增殖,直至大量死亡。细胞活性和生长曲线也显示,细胞在经历一个快速增殖期后,便进入平台期,此后细胞数量以及细胞活性都没有太大的起伏,而是在一定数值附近上下浮动。作为移植用干细胞,只有高的细胞活性和活率是不够的,还需要其保有干细胞特性和良好的生物安全性(保持正常核型)。因此,本文进而研究了经长期连续培养前后AD-MSCs的干细胞特性,并检测了一个月连续培养后细胞的核型。采用流式细胞仪和组织化学染色分析一个月连续培养前后细胞的干细胞特性；采用G显带染色体核型分析方法,观察经一个月连续培养后AD-MSCs的核型是否有异常。实验结果证实,AD-MSCs经一个月的连续不传代培养后,仍然保持着干细胞特性,包括：表达MSCs的表面标记蛋白、具有多向分化潜能,并且与培养前相比均没有显著性差异。G显带染色体核型分析则没有发现染色体的复制、倒位、易位、缺失或融合等异常现象,证实经一个月连续培养的AD-MSCs仍然具有稳定的核型,生物安全性可靠,可以应用于细胞治疗等领域。此外,本研究还探索了细胞周期分布以及细胞周期蛋白表达与AD-MSCs一个月连续生长增殖特性之间的关系。分别通过流式细胞仪技术和western blotting技术,检测了不同时间点细胞周期分布以及细胞周期蛋白A和细胞周期蛋白D1的表达。结果表明,细胞周期蛋白A的增量表达,可以促进AD-MSCs从S期向G2/M期过渡,使G2/M期细胞比率增加,处于分裂状态细胞含量增加,从而调控细胞进入快速增殖状态。而细胞周期蛋白D1的增量和减量表达,则可以促使S期的细胞比例增加和减少,从而调节细胞的生长增殖,使细胞进入增殖期和平台期。所以,综合以上结果可以得出,在AD-MSCs的长期连续培养过程中,细胞周期蛋白A和细胞周期蛋白D1通过它们的增量和减量表达,来调节细胞周期分布,从而影响着细胞的生长增殖。为了促进UC-MSCs和AD-MSCs向心肌的分化,本文采用生物活性因子与心肌诱导剂相结合的策略,研究了1-磷酸鞘氨醇(Sphingosine-1-phosphate, SIP)对UC-MSCs和AD-MSCs向心肌分化的影响,探索了其在心肌细胞培养液(CMCM)中适宜的作用时间和作用浓度。将UC-MSCs和AD-MSCs接种到培养板,用添加S1P的心肌细胞培养液或5-氮胞苷分化培养基诱导两种来源的间充质干细胞向心肌分化。采用免疫荧光染色检测心肌特异性蛋白,即α-肌动蛋白(a-actin)、缝隙连接蛋白(Connexin-43)以及肌球蛋白重链(MYH-6)的表达,并通过共聚焦显微镜和荧光显微镜进行观察；采用MTT法分析S1P分化培养基中细胞的活性变化；通过膜片钳检测分化细胞的钙瞬变(此为心肌细胞功能性指标)。结果表明,在CMCM诱导培养过程中,随着S1P浓度及其作用时间的增加,其促心肌分化作用增强,而细胞活性下降。因此,研究确定CMCM中S1P的适宜作用时间为14天,适宜作用浓度为0.5nmol/L。SIP与CMCM联合作用,不仅可以促进UC-MSCs和AD-MSCs表达心肌特异性蛋白,而且可以促进它们分化的细胞产生钙瞬变现象,即：具有类似心肌细胞的功能性。然而,S1P联合5-氮胞苷则仅仅是增强了分化细胞心肌特异性蛋白的表达,而不能促进分化细胞形成钙瞬变现象。其中的原因,可能与心肌细胞培养液中含有的生物活性因子(如转化生长因子)有关。更多还原

【Abstract】 Due to their multipotential, available in large numbers and easily accessible, human adipose-derived mesenchymal stem cells (AD-MSCs) and human umbilical cord mesenchymal stem cells (UC-MSCs) have become an attractive stem cell source for cell therapy and tissue regeneration. More than one month is needed for cell therapy, and time plays an important role in cell therapy. Further more, cell viability of MSCs in transplanted area is another key factor for improving cell therapy. However, bad transplanted environment (such as ischemia, oxidative stress, inflammatory reaction) and contact inhibition, which cause abundant death or apoptosis of MSCs, are unfavorable for application of cell therapy. Therefore, it is needed to find a suitable source of MSCs possessing more tolerance to the contact inhibition for improving the cell therapy. In addition, UC-MSCs and AD-MSCs have the potential to differentiate into cardiomyocytes and can express cardiomyocytes specific proteins, making them promising therapeutic candidates for treating damaged cardiac tissues. However, the differentiated cells induced from UC-MSCs or AD-MSCs by5-azacytidine or cardiomyocytes culture medium can hardly display functional characteristics similar to cardiomyocytes. Therefore, it is important to find a bioactive factor combining with cardiac differentiation medium for UC-MSCs or AD-MSCs to improve their functional cardiomyogenic differentiation.In this study, the growth and cell viability of AD-MSCs and UC-MSCs were investigated by CCK-8kits and hemocytometer everyday in30days of culture. Live/Dead staining and flow cytometry (FCM) were used to evaluate the cell viability of AD-MSCs and UC-MSCs during one month of culture. The results showed that AD-MSCs could be cultured up to the30th day in one passage while maintaining high level cell viability, and were seemed to be more tolerance to the contact inhibition. In addition, the cells displayed two plateau phases and three logarithmic phases during one month of culture. While UC-MSCs were not tolerance,to the contact inhibition, they couldn’t maintain high level cell viability in whole culture. Their cell viability significantly declined in later period of one month of culture. And the grow curves of UC-MSCs showed that they only displayed one logarithmic phase then went into the plateau phase. The cell numbers and cell viability of UC-MSCs kept mostly unchanged in the late of one month of culture. As a cell source of transplanting for clinical implication, the cells are required to not only maintain high cell viability, but also maintain their stem cell characteristics and normal chromosome type. The stem cell characteristics of AD-MSCs at the beginning and the end of culture were detected by flow cytometry (FCM) and histochemical staining. In addition, G banding karyotype analysis was used to evaluate biosecurity of AD-MSCs after one month of culture. The results showed that AD-MSCs could maintain their stem cell characteristics up to the end of one month of culture, including the capacity of mutilineage differentiation and the expression of hADSCs relative surface marker proteins. The results of G banding karyotype analysis showed that AD-MSCs after one month of culure still possessed a normal female chromosome type with no chromosome abnormalities being observed. This study demonstrates that AD-MSCs can be used as seed cells for application of cell therapy.In adition, to investigate the control factors of hADSCs growth during one month of culture, we detected the cell cycle distribution profiles by FCM using propidium iodide (PI) staining of DNA content, and the expression of cell cycle regulators including cyclin D1and cyclin A by western blotting. The results showed that increasing expression of cyclin A at protein level resulted in an increase in the percentage of AD-MSCs in the S and G2/M phases, promoting cells to go into logarithmic phases. Meanwhile, increasing or decreasing expression of cyclin D1at protein level caused a rise or decline in the proportion of AD-MSCs in the S phase, regulating cells to move into rapid proliferation or plateau phases. In conclusion, cyclin A and cyclin D1affect the distribution of cell cycle and regulate the growth of AD-MSCs.In this study, we have investigated the effects of sphingosine-1-phosphate (SIP) on cardiomyogenic differentiations of UC-MSCs and AD-MSCs in cardiomyocyte culture medium (CMCM) and5-azacytidine in vitro. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of respective combinations of SlP-5-azacytidine and S1P-CMCM on cell viability were evaluated by methyl thiazolyl tetrazolium (MTT) assays. Functional characteristics similar to cardiomyocytes were evaluated through detecting calcium transient. The results showed that the differentiations towards cardiomyocytes of UC-MSCs or AD-MSCs in CMCM were enhanced with SIP concentration increasing, but cell activities declined. Therefore, our data showed that the suitable differentiation time was around14days, and the optimal concentration of SIP was0.5μmol/L in CMCM differential medium. Moreover, S1P working together with CMCM can not only enhance the differentiations of UC-MSCs or AD-MSCs into cardiaomyocytes, but also promote their differentiations towards functional cardiomyocytes, generating the calcium transients from the induced cardiomyocytes. Wherase SIP with5-azacytidine only enhanced the expressions of the three cardiac specific proteins but could not give rise to the specific electrophysiological properties (the calcium transient) in the committed UC-MSCs or AD-MSCs. This difference could be due to some cellular stimulus (e.g. transforming growth factor-beta, TGF-β) existing in CMCM but not in5-azacytidine induction medium.更多还原