RS细胞血清剥夺体外模型的建立及雌激素保护机制的研究

发布时间：2018-07-03 20:16

本文选题：骨髓间充质干细胞 + RS细胞　；参考：《上海交通大学》2009年硕士论文

【摘要】： 近年来心肌再生过程和技术的相关研究已经成为再生医学和心脏病学研究的热点,骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs)作为细胞治疗最常用候选种子细胞之一,由于具有分化成多种非造血组织的能力而成为该领域研究的焦点。但干细胞移植入缺血性心脏后存活率较低的问题却成为该技术应用的瓶颈。因此阐明移植后干细胞凋亡的分子机制并改善局部微环境,降低因缺血引起的细胞死亡及凋亡已成为心脏再生医学研究的重要课题。有研究表明雌激素在缺血性损伤后的心肌重塑过程中发挥重要作用,但目前体外雌激素对心肌缺血引起的MSC坏死和凋亡是否具有保护作用国内外尚未见报导。本研究的目的正在于探讨雌激素在缺血环境中对MSCs的保护作用及作用机制。快速自我更新细胞(rapidly self-renewing cells,RS细胞)是在探索获取具有更强自我更新能力及多向分化潜能均质骨髓间充质干/祖细胞群过程中发现的一个小细胞亚群,该类细胞表达与成熟MSCs(mature MSCs,mMSCs)不同的蛋白及表面抗原,用于体内长期移植或细胞治疗更具明显优势。因此,以RS细胞作为本研究的研究对象建立体外血清剥夺模型模拟心肌缺血环境,一方面进一步推进RS细胞生理机制的研究并完善其临床应用,另一方面为探讨雌激素作用机制提供新的思路。本实验分别以大鼠骨髓间充质干细胞rMSCs中的mMSCs和RS细胞亚群为研究对象,探讨血清剥夺体外培养模拟心脏缺血环境对不同MSC亚群细胞活性的影响及雌激素的保护作用。本文的创新点在于第一次使用雌激素作为心脏缺血环境中MSCs的保护剂,分析其保护作用能力,并初步探索其作用机制。此外,着重以RS细胞为研究对象,获得高质量富含RS细胞的rMSC样本,以期加快其临床应用进程。本实验分别以3-1000个细胞/cm2接种rMSCs,培养7-14天,期间通过倒置显微镜观察细胞形态并进行图像分析,测定生长曲线并进行生长动力学分析,以及通过CFU测定评估细胞增殖潜能。结果表明,10个细胞/cm2为最佳接种密度,所获得rMSC样本在培养7-10天时细胞增殖能力较强。并且发现随着接种密度的升高,rMSC增殖能力下降的越快。此外,mMSC对RS细胞增殖产生抑制作用,可能同时通过接触抑制和分泌某些细胞因子。获得高质量富含RS细胞的rMSC样本后,本实验以无血清体外培养rMSCs为模型,经细胞形态学观察、Hoechest33342荧光染色、Annexin V-PI双染流式细胞仪检测和PI标记细胞周期分析,观察细胞形态变化、核变化、活性/凋亡率变化和对细胞周期的影响。结果显示SD 12小时后rMSC皱缩变圆、染色质固缩,出现凋亡各阶段形态特征;与对照组相比mMSC及RS活性明显降低并且凋亡率升高(P均小于0.01),与血清剥夺组相比添加10nM E2后细胞活性显著提高(P0.01);并且RS细胞亚群对SD环境表现出更强的耐受性,与mMSC亚群相比存活率提高2.75倍,凋亡率降低近三分之一,并在雌激素的保护作用下继续保持良好增殖。同时还发现SD环境使细胞于S期受阻,E2则可以使这一比例降低5.8%。上述结果表明雌激素可以提高干细胞自我保护能力,并且RS细胞具有更强的自我保护能力和自我调节机制。此外,推测雌激素在血清剥夺条件下对MSC的保护作用可能包括通过核受体调控细胞周期,以及与细胞表面受体等非转录途径有关。
[Abstract]:Bone marrow mesenchymal stem cells ( MSCs ) , as one of the most commonly used candidate seed cells for cell therapy , has become the focus of the research in the field of regenerative medicine and cardiologist . However , the problem of low survival rate of bone marrow mesenchymal stem cells ( MSCs ) has become the bottleneck in the field .

It has been shown that estrogen plays an important role in the process of myocardial remodeling after ischemic injury , but the effect of estrogen on myocardial ischemia - induced MSC necrosis and apoptosis has not been reported at home and abroad . The purpose of this study is to investigate the protective effect and mechanism of estrogen on MSCs in ischemic environment .

Rapid self - renewal cells ( RS cells ) are a small subset of cells found in mesenchymal stem / progenitor cells with stronger self - renewal ability and multi - directional differentiation potential . These cells express different proteins and surface antigens than mature MSCs ( mature MSCs and mMSCs ) and are used in vivo long - term transplantation or cell therapy . Therefore , using RS cells as the research object of this study establishes an in vitro serum deprivation model to simulate the myocardial ischemia environment . On the one hand , the study of the physiological mechanism of RS cells is further advanced and its clinical application is improved . On the other hand , new ideas are provided to explore the mechanism of estrogen action .

In this experiment , the mMSCs and RS cell subsets of rat bone marrow mesenchymal stem cells ( rMSCs ) were studied . The effects of serum deprivation on the activity of different MSC subgroups and the protective effects of estrogen were discussed . The innovative point of this paper was to use estrogen as the protective agent of MSCs in the ischemic environment of the heart for the first time , to analyze its protective effect and to explore its mechanism of action . In addition , the rMSC samples with high quality RS - rich cells were obtained with RS cells as the research subjects , with a view to accelerating its clinical application process .

In this experiment , rMSCs were seeded with 3 - 1000 cells / cm2 and cultured for 7 - 14 days . The cell morphology was observed by an inverted microscope . The growth curve was measured and the growth kinetics of the cells was evaluated . The results showed that 10 cells / cm2 were the best seeded density , the faster the cell proliferation ability was obtained with the increase of the inoculation density , the faster the rMSC proliferation ability decreased . In addition , the mMSC inhibited the proliferation of RS cells , which could inhibit and secrete some cytokines at the same time .

The results showed that estrogen can improve the self - protection ability of the stem cells and increase the apoptosis rate ( P < 0.01 ) .
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【共引文献】