【作者】 林先和；

【导师】 李隆贵；

【作者基本信息】 第三军医大学 ， 内科学， 2004， 博士

【摘要】 研究背景： 心肌细胞在出生后就不能再生，对有丝分裂信号的反应是细胞肥大而不是增生。心肌细胞的丧失会导致所在区域心肌收缩功能减退，以致心力衰竭，坏死心肌终而被成纤维细胞形成的疤痕组织所取代。近年来，采用自体细胞和生长因子修复组织的再生医学迅猛发展。有人将培养的心肌细胞移植到损伤区心肌内能够明显改善心功能，并称之为“细胞心肌成形术”(cellular cardiomyoplasty)，有可能成为治疗心力衰竭的有效手段。许多作者对胚胎干细胞诱导成心肌细胞进行了大量研究，并取得了显著进展。但是，胚胎干细胞移植仍然存在许多问题，诸如免疫排斥，伦理道德问题等。继胚胎干细胞后，骨髓间充质干细胞(MSC)受到了高度重视。来源于中胚层的MSC，是多能干细胞，在诱导后可以定向分化为成骨细胞、软骨细胞、脂肪细胞、成肌细胞以及肌腱细胞等多种细胞。MSC与心肌细胞同属于中胚层。近期已经有人应用5-氮杂胞苷(5-Aza)成功地将体外培养的MSC诱导成为心肌样细胞。Tomita等将人骨髓MSC植入免疫缺陷鼠心肌后，发现这些细胞可分化成为心肌细胞，且表达心肌细胞特有的desmin,α-actin，β-MHC2。有作者将骨髓MSC注射入心梗边缘部位心肌内，这些移植的MSC分化成为心肌细胞，且受损的心功能得到明显改善。进一步研究表明，MSC经5-Aza诱导后分化为心肌样细胞再行移植受损心肌部位，疗效明显优于未诱导组。目前对于骨髓MSC定向分化为心肌样细胞的研究处于起步阶段，心肌样细胞定向分化率仍然不高，对于心肌分化的调控机制研究甚少。因此，深入研究MSC分化过程中的调控机制对于寻求控制干细胞分化方向的手段，从而提高MSC向心肌样细胞定向分化率，促进细胞心肌成形术的应用有重要意义。 PPARγ(peroxisome proliferator-activated receptorγ)作为核激素受体超家簇中的成员，是细胞分化重要的调控因子。通过调节其他转录因子的表达，参与调节MSC分化，并在诱导MSC分化不同特异性表型之间起到开关作用。PPARγ在MSC向心肌样细胞分化过程中的作用如何，目前尚无相关报道。 资料显示，Nkx 2.5、GATA4、MEF-2C等基因是调控心肌细胞分化开始阶段的重第三军医大学博士学位论文要转录因子。本文应用培养小鼠MSC为研究材料，以5一Aza作为诱导剂，叫睬美辛作为PPARY的配体，利用表达载体pEGFP一1一PPA助2转染MsC，检测PPA助对细胞分化及其对转录因子MEF一ZC、Nkx2.5、GATA4 mRNA表达的影响，旨在探讨PPA助对MSC分化为心肌样细胞的影响及其在分化过程中的调控作用。 方法与结果: 第一部分:探讨了小鼠MSC的体外分离、纯化、扩增、多向诱导和向心肌样细胞定向分化的诱导条件。首先采用密度为1.082留ml的Percou细胞分离液分离小鼠骨髓细胞中的单个核细胞，所获细胞应用DME树低糖培养基+l0%胎牛血清进行培养，通过及时、反复传代对MSC进行纯化和扩增。平均5一6d在细胞达到80%左右融合时进行传代。采用流式细胞仪检测细胞周期。应用不同的诱导方法对细胞进行多向诱导分化，使细胞向成骨细胞、成心肌样细胞、成脂肪细胞三个不同的方向分化。诱导成心肌样细胞分化时选取了5一Aza3林mol/L、5林mol/L、10林mol/L三种不同浓度。应用四环素荧光标记检测钙结节，油红O染色检测脂滴，免疫组织化学检测ThT表达。 结果:MSC细胞形态保持均一的短梭形。12一14d细胞长成单层。细胞生长曲线:第1、Zd细胞数量无明显增加，第3d开始细胞数量明显增加，MSC在第7d达到最高峰。第8d开始进入平台期。细胞周期结果显示:MSC中GI期的细胞占91 .74%，S+GZ的细胞约占8.26%，表明只有少数细胞进入了活跃增殖期。成骨诱导后，细胞出现钙结节。成脂诱导的细胞胞浆内出现大量脂滴。成心肌样细胞诱导中以5-Aza3林mol几浓度为最佳，诱导后l月，部分细胞TnT表达阳性。5一Aza以5林mol/L及10娜ol/L浓度诱导后虽有肌管样结构出现，但TnT表达始终呈阴性。 第二部分:应用叫噪美辛作为P队RY的配体作用于MSC以激活PPA助。将细胞分为4组:A组用5一Aza3林mol/L诱导细胞分化;B组分别采用叼}噪美辛10一、10一，mol/L两种不同浓度作用于MSC，观察细胞生长情况，形态变化;C组联用5一Aza鸿}噪美辛作用于MsC;D组为对照组，不诱导。应用油红一O检测脂滴，免疫组织化学检测PPARY表达情况。 结果:叫噪美辛在10一mol几浓度时对细胞有明显的毒性，而10一smol/L时细胞生长良好。对照组及单用10一，mol几叫睬美辛作用Msc时，PPA助表达均呈阴性。单用叫垛美辛并不能诱导MSC分化。单用5一Aza3卜mol几诱导后的第3d，部分细胞PPARY表达阳性。联用6一Aza/叫噪美辛诱导时发现，诱导后第3d几乎所有细胞PPA助表达阳性，继续培养后分化为脂肪细胞。第三军医大学博士学位论文 第三部分:分题卜重组表达载体pCAG一PPARYZ及pEGFP一Nl。将前者中的PPA助2目的片段构建入后者中。采用5’端一粘性末端一3’端平端构建策略对目的基因进行亚克隆，构建成pEGFP一Nl一PPA助2表达载体。酶切鉴定与测序鉴定相结合。分题2:经鉴定正确的重组质粒用脂质体转染法转染入MSC，应用吼;:并采用休克法进行筛选?更多还原

【Abstract】 Background and Objectives Cardiomyocytes do not regenerate after birth, and they respond to mitotic signals by cell hypertrophy rather than by cell hyperplasia. Loss of cardiomyocytes leads to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissues. The regenerative medicine which repaires damage tissue with autologous cell transplantation has been developed recent years. Studies revealed that transplantation of cultured cardiomyocytes into the damaged myocardium could prevent postinfarction heart failure. This new way which was called cellular cardiomyoplasty has been proposed as a future method for the treatment of heart failure. Many achievements have been gained in the feild of inducing the differentiation of embryonic stem cells into cardiomyocytes. But there are still problems we didn’t solved, such as immunologic rejection and ethnics. Recent published reports have revealed that mesenchymal stem cells (MSCs) from mesoderm are multipotential stem cells. MSCs induced with 5-Aza can differentiate into cardiac-like muscle cells in culture and in vivo in ventricular scar tissue and improve myocardial function. In addition, the cells are easy to be collected and cultured, and have the potential ability to expansion. Unfortunately, only about 30% MSCs induced with 5-Aza can differentiate into cardiomyocytes, and little is known about the molecular mechanism.PPARs (peroxisome proliferator activated receptor) are a family of ligand-activated nuclear hormone receptors which include three sub-types, PPARa, PPARP and PPARr. It was reported that activation of PPARy is involved in cell proliferation, differentiation and apoptosis. But its certain role and regular mechanism in the differentiation of MSCs into cardiomyocytes is to be determined. The current study is initiated to investigate the regulate mechanism of PPARr and its ligand indomethacin in the differentiation of mouse MSCs into cardiomyocytes in vitro.Materials and Methods We examined the isolation, purification, expansion of mouse MSCs and capacity to differentiate into cardiomyocytes in vitro. First, bone marrowmononuclear cells were harvested from male Balb/c mouses femur and tibia with Percoll separating medium at the density of 1.082g/mL. Then, cells were cultured with DMEM/ Low glucose medium and 10% foetus cattle blood serum. Mesenchymal stem cells were obtained by removing the non-adherent cells 24h after seeding and purified and expanded through passaging in time. Primary cells were passgaed at a ratio of one to three plates when they reached 70-80% confluence. After 2-3 passages, cells were induced to differentiate into three different directions, such as osteoblasts, lipoblast and cardiomyogenesis, with three different inducing methods. Different concentrations of 5-Aza were selected including 3, 5 and 10umol/L. Cell cycles were measured with flow cytometry.To examine the effects of PPARy on the differentiation of mouse MSCs into cardiomyocytes, we constructed the mammalian expression plasmid pEGFP-N1-PPARr2 for mouse PPARy2. Indomethacin was choosed as ligand to activate PPARy. Cells were randomly divided into groups as follows: Control group; 5-Aza group (induced with 5-Aza); indomethacin group (induced with indomethacin); 5-Aza/indomethacin group(induced with 5-Aza/indomethacin); PPARr2 transfection group (transfected with expression plasmid pEGFP-N1- PPARr2 and activated with indomethacin). Cells were transfected using the cationic liposome-mediated transfection method and incubated under conditions permissive for differentiation in the presence of indomethacin. Cardiomyogenesis was monitored by immunostaining with an antibody, Troponin T, directed against the troponin. Adipogenesis was measured as lipid droplet by staining with oil red O. Osteogenesis was measured as calcium nod and by fluorescence staining with tetracyn. The changes of PPARy,GATA4, MEF-2C and Nkx2.5 mRNA expression were observed with RT-PCR at the time of lh,3h,5h,1更多还原