【作者】 陈炜；

【导师】 张雷；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2012， 博士

【摘要】 目的：心血管疾病是目前世界上危害人类健康的主要疾患之一，具有极高的发病率。一旦发生急性心肌梗塞（acute myocardial infarction, AMI）或其他心脏病等，会造成功能性心肌细胞数量下降，瘢痕组织形成，心室发生重构，从而导致心功能下降，甚至最终诱发心力衰竭而危及生命。采用细胞移植，将外源性的有功能细胞移植到梗死心肌，发挥其综合生物学作用，从而改善心脏功能，这是个具有极大潜力的治疗方法。在过去几十年中，人类为寻找一种能够修复损伤心肌、恢复心功能的干细胞投入了极大的热情和努力，目前研究最多的骨髓间充质干细胞（bone marrowmesenchymal stem cells, BMSCs）属于成体干细胞（adult stem cells, ASCs），与其他干细胞相比具有明显的优势：①可以自体移植，不涉及伦理道德问题。②易于分离，体外培养具有高度的扩增能力，并且在体外多次传代后基因稳定性良好。③具有多向分化潜能。④具有低免疫原性和可移植性。因此，BMSCs是用于心血管疾病干细胞移植治疗的较理想的种子细胞。BMSCs体外向心肌细胞分化的方法大致有三种：①药物诱导；②与心肌细胞共培养；③基因修饰。然而诱导效率低仍是目前存在的问题，如何能够提高诱导分化的效率成为科研人员研究的热点。对BMSCs进行基因修饰，从分子水平促进BMSCs的心肌细胞分化，是近几年随着分子生物学技术发展而新兴的诱导方法，旨在通过启动某个或某些关键基因，激活心肌分化基因调控网络，实现BMSCs向心肌细胞分化的目的。心脏早期转录因子在心脏发育早期表达，其中最主要的有Nkx2.5、GATA4、TBX5等，它们调控许多心脏结构蛋白基因的表达，对心脏正常发育具有十分重要的作用，曾被作为外源基因导入胚胎干细胞等，促进其向心肌细胞方向分化。用于MSCs向心肌细胞分化的药物诱导剂，文献报道有5-氮胞苷（5-azacytidine,5-aza）、二甲基亚砜（dimethylsulfoxide, DMSO）、胰岛素、地塞米松、抗坏血酸等。其中，5-aza是目前最常用的诱导剂，但作为肿瘤治疗药物也存在细胞毒性大等不利于临床应用的问题。近年我们研究了一种人体激素——催产素（Oxytocin, OT）对干细胞向心肌细胞分化的诱导能力。研究发现，在心血管方面，OT除了具有降压等调节作用外，对于心脏的发生也具有一定影响。并且能够诱导P19细胞、P19CL6细胞、胚胎干细胞、心脏来源的Sca-1阳性细胞、心旁细胞等多种细胞向心肌细胞分化。本实验采用心脏早期转录因子Nkx2.5、GATA4、TBX5转染BMSCs、诱导剂OT诱导或两种方法联合应用，探讨Nkx2.5、GATA4、TBX5对BMSCs向心肌细胞分化的作用和影响，为实现BMSCs高效心肌细胞分化提供实验依据，为干细胞移植修复心肌损伤提供良好的细胞来源。方法：1骨髓间充质干细胞的分离、培养和鉴定采用贴壁分离筛选法与换液、严格控制消化时间的传代方法结合，分离、纯化、扩增培养SD大鼠BMSCs。从三个方面对分离扩增培养的BMSCs进行鉴定：（1）观察培养细胞的黏附生长特点；（2）流式细胞技术联合检测细胞表面分子CD90、CD29、CD45的表达情况；（3）体外诱导细胞向脂肪细胞、骨细胞、软骨细胞分化，检测其多向分化潜能。2外源基因Nkx2.5、GATA4、TBX5诱导骨髓间充质干细胞向心肌细胞分化实验分组：A1组（转染pEGFP-N1-Nkx2.5）、A2组（转染pEGFP-N1空质粒）、A3组（空白组）；B1组（转染pVP22-GATA-4/myc-His）、B2组（转染pcDNA3.1空质粒）、B3组（空白组）；C1组（转染pcDNA3.1-TBX5）、C2组（转染pcDNA3.1空质粒）、C3组（空白组）。转染前通过预实验确定最佳细胞转染密度、最佳转染体系，采用阳离子转染试剂Lipofectamine2000转染各组BMSCs。转染后48h经Western blot检测各组外源基因表达。转染后继续培养4w，免疫细胞化学、Western blot检测各组细胞心肌肌钙蛋白T（cardiac troponin T, cTnT）、连接蛋白43（connexin43,Cx43）的表达。分别取P3BMSCs和转染组转染后继续培养1w、2w、3w、4w的细胞标本，Real Time PCR检测心肌相关基因Cx43、β-肌球蛋白重链（β-myosin heavy chain, β-MHC）、肌球蛋白轻链-2（myosin light chain-2,MLC-2）的表达。3外源基因Nkx2.5、GATA4、TBX5联合催产素诱导骨髓间充质干细胞向心肌细胞分化实验分组：①转染组：又分为Nkx2.5组（转染pEGFP-N1-Nkx2.5）、GATA4组（转染pVP22-GATA-4/myc-His）和TBX5组（转染pcDNA3.1-TBX5）；②转染+药物诱导组：又分为Nkx2.5+OT组（转染pEGFP-N1-Nkx2.5+OT诱导）、 GATA4+OT组（转染pVP22-GATA-4/myc-His+OT诱导）、TBX5+OT组（转染pcDNA3.1-TBX5+OT诱导）；③单纯药物诱导组（OT诱导组）；④空白组。采用阳离子转染试剂Lipofectamine2000转染各组BMSCs。转染后继续培养4w，免疫细胞化学、Western blot检测各组细胞cTnT、Cx43的表达；透射电镜技术检测各组细胞超微结构变化。结果：1骨髓间充质干细胞的分离、培养和鉴定分离原代细胞48h首次换液后，贴壁细胞呈圆形、多角形、梭形，细胞生长缓慢。3～4d后贴壁细胞分裂增殖迅速，相邻细胞逐渐相互汇合，9～11d细胞汇合可达80%，呈旋涡状或放射状排列。传代后BMSCs贴壁比原代细胞快，细胞呈均匀分布生长。随着细胞换液和传代，贴壁细胞形态逐渐趋于一致；传代至第3代时BMSCs形态基本单一，呈梭形。流式细胞仪检测生长良好的P3细胞中CD90+/CD29+/CD45-细胞达99%以上，说明获得的BMSCs具有较高的均一性，细胞纯度较高。BMSCs成脂诱导1w后，部分细胞内有数量较多的小脂滴积聚，以后细胞中的脂滴变大、增多，含有大量脂滴的脂肪样细胞逐渐增多；2w后，用脂肪特异性结合染料油红O染色检测，细胞胞质中的脂滴被染为橘红色。BMSCs成骨诱导后，细胞逐渐转变为多角形或不规则形，细胞逐渐汇合，排列紧密，呈铺路石状；2w后，用可以和钙发生显色反应的茜素红进行细胞化学染色，细胞外基质中有被染成红色的钙化点。BMSCs成软骨诱导后，局部部分细胞逐渐转变为圆形或椭圆形，并且增殖迅速，聚集生长；2w后，局部形成多个结节样结构，用可将硫酸化蛋白聚糖染为蓝色的阿尔新蓝染色检测，将软骨结节样结构染成蓝色。2外源基因Nkx2.5、GATA4、TBX5诱导骨髓间充质干细胞向心肌细胞分化通过预实验发现：以4×10~4/cm~2密度传代，在次日转染时达到90%～95%汇合；质粒DNA（μg）和转染试剂Lipofectamine2000(μl)的比率为1:2.5，此条件下转染可取得较好的效果。转染48h后，Western blot检测A1、A2、A3各组Nkx2.5:EGFP融合蛋白表达，结果表明转染组A1组有外源性Nkx2.5表达，而空质粒组A2组、空白组A3组均无表达；检测B1、B2、B3各组myc蛋白表达，结果表明转染组B1组有外源性GATA4表达，而空质粒组B2组、空白组B3组均无表达；检测C1、C2、C3各组TBX5蛋白表达，结果表明三组均有TBX5表达，其中转染组C1组比空质粒组C2组、空白组C3组TBX5表达显著增高（P<0.05）。证明脂质体能够成功介导质粒pEGFP-N1-Nkx2.5、pVP22-GATA-4/myc-His、pcDNA3.1-TBX5转染BMSCs。外源基因转染培养4w后形态学观察，A1组、B1组、C1组细胞呈长梭形，部分细胞增大、增宽，随着培养时间延长，局部细胞密集重叠生长，当细胞密度达到一定程度后，细胞不再增殖，各组细胞形态改变相似；A2组、A3组、B2组、B3组和C2组、C3组细胞也有局部少量细胞密集重叠生长。外源基因转染培养4w后，免疫细胞化学检测结果表明：A1组、B1组、C1组部分细胞呈cTnT阳性，cTnT阳性细胞的胞质中可见棕黄色丝网状及颗粒样结构，以局部密集重叠生长部及放射周边的细胞中cTnT呈强阳性。A1组、B1组、C1组部分细胞呈Cx43阳性，Cx43阳性细胞的胞质中可见棕褐色颗粒，以局部密集重叠生长部及放射周边的细胞中Cx43呈强阳性。A2组、A3组、B2组、B3组和C2组、C3组只有少量细胞cTnT、Cx43表达阳性。各组进行积分吸光度（IA）值统计结果显示：A1组、A2组、A3组比较，转染组A1组cTnT、Cx43表达最高，空质粒A2组、空白组A3组cTnT、Cx43表达较低，与A1组相比均有显著差异（P<0.05）；B1组、B2组、B3组比较，转染组B1组cTnT、Cx43表达最高，空质粒B2组、空白组B3组cTnT、Cx43表达较低，与B1组相比均有显著差异（P<0.05）；C1组、C2组、C3组比较，转染组C1组cTnT、Cx43表达最高，空质粒C2组、空白组C3组cTnT、Cx43表达较低，与C1组相比均有显著差异（P<0.05）。外源基因转染培养4w后，Western blot结果显示转染组cTnT蛋白呈明显高表达，空质粒组、空白组几乎无表达。统计结果与免疫细胞化学cTnT检测结果一致。Real Time PCR结果显示： A1、B1、C1组Cx43表达水平趋势变化相似，均于第1w开始逐渐增高，第2w或3w时表达最高，之后表达回落。β-MHC在A1组表达水平于第1w至第3w时显著高于P3BMSCs（P<0.05），第4w时表达减弱，与P3BMSCs比较无差异（P>0.05）；B1组β-MHC表达水平第1w、第2w时与P3BMSCs间均无显著差异（P>0.05），第3w时表达显著增高，与P3BMSCs有显著差异（P<0.05），第4w时回落，与P3BMSCs无显著差异（P>0.05）；β-MHC在C1组表达水平从第1w开始呈逐渐增高趋势，第4w时表达最高，但前3w与P3BMSCs组比较无差异（P>0.05），第4w时表达与P3BMSCs比较有显著差异（P<0.05）。MLC-2表达水平在A1、C1组第1w时与P3BMSCs比较无显著差异（P>0.05），第2w时表达显著增高，至第3w时逐渐回落，与P3BMSCs比较有显著差异（P<0.05），第4w时表达减弱；B1组MLC-2表达水平第1w、2w时与P3BMSCs比较均无显著差异（P>0.05），第3w时显著增高，与P3BMSCs有显著差异（P<0.05），第4w时回落，与P3BMSCs间比较无显著差异（P>0.05）。3外源基因Nkx2.5、GATA4、TBX5联合催产素诱导骨髓间充质干细胞向心肌细胞分化外源基因转染培养4w后，倒置显微镜形态学观察发现，转染组、转染+药物诱导组及OT诱导组细胞均呈长梭形，部分细胞增大、增宽，随着培养时间延长，局部细胞密集重叠生长，当细胞密度达到一定程度后，细胞不再增殖，各组细胞形态改变相似；空白组细胞也有局部少量细胞密集重叠生长。外源基因转染培养4w后，免疫细胞化学检测cTnT、Cx43表达结果显示，转染组（Nkx2.5组、GATA4组、TBX5组）、转染+药物诱导组（Nkx2.5+OT组、GATA4+OT组、TBX5+OT组）、OT诱导组部分细胞呈cTnT阳性，cTnT阳性细胞的胞质中可见棕黄色丝网状及颗粒样结构，以局部密集重叠生长部及放射周边的细胞中cTnT呈强阳性。转染组、转染+药物诱导组、OT诱导组部分细胞呈Cx43阳性，Cx43阳性细胞的胞质中可见棕褐色颗粒，以局部密集重叠生长部及放射周边的细胞中Cx43呈强阳性。空白组只有少量细胞cTnT、Cx43表达阳性。各组积分吸光度（IA）值统计结果显示：转染组(Nkx2.5组、GATA4组、TBX5组)、转染+药物诱导组（Nkx2.5+OT组、GATA4+OT组、TBX5+OT组）、OT诱导组、空白组比较，cTnT在转染组呈明显的高表达，转染+药物诱导组表达最高，OT诱导组也有较高表达，空白组仅有少量表达，各诱导组与空白组之间比较均有显著差异（P<0.05）；Cx43表达水平在转染组、转染+药物诱导组均较高， OT诱导组也有明显表达，空白组仅有少量表达，各诱导组与空白组之间比较均有显著差异（P<0.05）。外源基因转染培养4w后，Western blot结果显示：转染+药物诱导组cTnT蛋白表达最高，转染组呈明显的高表达，OT诱导组也有较明显表达，空白组几乎无表达，各诱导组与空白组之间比较均有显著差异（P<0.05）。统计结果与免疫细胞化学cTnT检测结果一致。透射电镜下可见未转染的P3BMSCs和空白组细胞呈长梭形，细胞质中细胞器较丰富，可见较多核糖体及线粒体、粗面内质网、溶酶体等，少数细胞间偶见缝隙连接。转染组、转染+药物诱导组和OT诱导组细胞呈梭形，细胞核呈卵圆形，较大，位于细胞中央，细胞质中细胞器丰富，可见较多线粒体、核糖体、粗面内质网、溶酶体等；细胞核周的细胞质内出现一些平行排列的肌丝样结构，部分细胞周边突起和细胞末端可见大量密集排列的肌丝样结构，隐约可见电子密度高与电子密度低区域交错排列；细胞侧面或细胞末端可见较多明显的缝隙连接。其中，转染组和转染+药物诱导组细胞内肌丝样结构和缝隙连接更多、更明显，OT诱导组细胞内肌丝样结构和缝隙连接相对较少。结论：1成功分离、扩增及鉴定了SD大鼠BMSCs。2应用阳离子脂质体转染试剂Lipofectamine2000介导质粒pEGFP-N1-Nkx2.5、pVP22-GATA-4/myc-His、pcDNA3.1-TBX5成功转染入SD大鼠BMSCs。3SD大鼠BMSCs分别转染心脏早期转录因子Nkx2.5、GATA4、TBX5后，能够促进BMSCs向心肌细胞分化，在基因和蛋白水平表达心肌源性特异标志物cTnT、Cx43、β-MHC、MLC-2，具有发育早期的心肌细胞结构特点。4OT作为诱导剂能够促进SD大鼠BMSCs向心肌细胞分化，具有发育早期心肌细胞结构特点。5心脏早期转录因子转染+OT诱导联合应用，有促进BMSCs向心肌细胞方向分化的叠加效应，cTnT蛋白表达水平有较明显增高，但Cx43蛋白表达没有明显变化。更多还原

【Abstract】 Objective: Cardiovascular disease, with a high incidence, is greatlyharmful to human health. In acute myocardial infarction （AMI）, the loss offunctional myocardial cells and the formation of scar tissue lead to ventricularreconstruction, even heart failure and life-threatening. It is a very promisingtreatment to transplant exogenous functional cells into infarcted myocardiumto improve cardiac function. In the past decades, many kinds of stem cellswere tried as cell sources for cell transplantation therapy. Bone marrowmesenchymal stem cell （BMSC） is one of the best candidates. BMSCs,classified in adult stem cells （ASCs）, have obvious advantages compared withother stem cells:①They can be used for autologous transplantation, withoutinvolving ethical controversy.②They can be isolated easily, then amplifyhighly in vitro. They are genetic stable for several passages in vitro.③Theyhave multilineage differentiation potential.④They have low-immunogenicityand portability. So, BMSCs are ideal seed cells for stem cell transplantationtherapy for cardiovascular disease.There are three methods to induce BMSCs to differentiate intomyocardial cells in vitro:①be induced by drug;②be co-cultured withmyocardial cells;③be modified genetically. However, the differentiationefficiency is still low and how to improve it is becomming the research focus.With the development of molecular biology techniques, it is a newmethod to modify BMSCs genetically to promote the cardiomyocytedifferentiation. It is aimed to start one or some of the key genes and activategene regulatory networks of cardiac differentiation. Cardiac-specific earlytranscription factors, such as Nkx2.5, GATA4, TBX5, regulate geneexpression of cardiac structure and play an important role in normal development of the heart. They have been transfected into embryonic stemcells （ESCs） to promote the cardiomyocyte differentiation.The drug inducers for MSCs cardiomyocyte differentiation include5-azacytidine （5-aza）, dimethylsulfoxide （DMSO）, the combination of insulin,dexamethasone, ascorbic acid. The commonly used agent,5-aza, is notsuitable for clinical treatment because of the cell toxicity. In recent years, wehave researched the possibility of oxytocin （OT） as a drug inducer of stemcell’s cardiomyocyte differentiation. Researches show that in addition of theability to regulate blood pressure, OT plays a role on heart development. OThas been used to induce P19cells, P19CL6cells, ESCs, adult cardiacSca-1-positive cells and cardiac side population cells into cardiomyocytes.Therefore, in this study we try to transfect BMSCs with Nkx2.5, GATA4,TBX5, or use OT as the inducer, or use the combination of two methods. Theaim is to explore the relationship between Nkx2.5, GATA4, TBX5andcardiomyocyte differentiation of BMSCs, and to provide effective strategy fortransforming various BMSCs into myocardial cells.Methods:1The isolation, culture, and characterization of BMSCsSD rat BMSCs were isolated by the whole bone marrow culture,amplified by serial subcultivation and purified by digestion time control andsubcultivation. We characterized BMSCs in three aspects:①the adheredcultured cells were observed;②the expression of CD90, CD29, CD45weredetected by flow cytometry;③the multilineage differentiation potential wasdetected by adipogenic, osteoblasts and chondrogenic differentiation in vitro.2Exogenous genes of Nkx2.5, GATA4, TBX5induced BMSCs todifferentiate into myocardial cells.Experimental groups: A1group （transfected with pEGFP-N1-Nkx2.5）,A2group （transfected with pEGFP-N1empty plasmid）, A3group （blankgroup）; B1group （transfected with pVP22-GATA-4/myc-His）, B2group（transfected with pcDNA3.1empty plasmid）, B3group （blank group）; C1group （transfected with pcDNA3.1-TBX5）, C2group （transfected with pcDNA3.1empty plasmid）, C3group （blank group）. Before transfection,preliminary experiment was used to determine the suitable cell seeding densityand the best transfection system. Using the cationic liposome reagent,Lipofectamine2000, plasmid was transfected into BMSCs. Exogenous geneexpression were detected with western blot after48hours of transfection.After4weeks of transfection and the following culture, the expression ofcardiac troponins T （cTnT） and connexin43（Cx43） were detected withimmunocytochemical staining and Western blot. Real Time PCR was used todetect the expression of Cx43, β-myosin heavy chain （β-MHC）, myosin lightchain-2（MLC-2） in P3BMSCs and experimental groups.3Combination of exogenous genes of Nkx2.5, GATA4, TBX5and oxytocininduced BMSCs into myocardial cells.Experimental groups:①transfection groups: Nkx2.5group （transfectedwith pEGFP-N1-Nkx2.5）, GATA4group （transfected withpVP22-GATA-4/myc-His）, TBX5group （transfected withpcDNA3.1-TBX5）;②transfection+induced drug groups: Nkx2.5+OT group（transfected with pEGFP-N1-Nkx2.5+OT induction）, GATA4+OT group（transfected with pVP22-GATA-4/myc-His+OT induction）, TBX5+OT group（transfected with pcDNA3.1-TBX5+OT induction）;③OT group （OTinduction）;④blank group. Using Lipofectamine2000, plasmid wastransfected into BMSCs. After4weeks of transfection and the followingculture, the expression of cTnT and Cx43were detected withimmunocytochemical staining and Western blot, and the ultrastructuralchanges of the cells were observed.Results:1The isolation, culture, and characterization of BMSCsThe first time to exchange the medium of primary cells was at48hoursafter islation. The adherent cells were round or polygonal or spindle-shaped.After3to4days, primary cells began to proliferate rapidly. After9to11days,cells arranged swirlingly or radially, reaching80%confluence. Passage cellsgrew rapidly. With medium exchange and passage, the morphology of adherent cells was becoming identical. The third passage cells werespindle-shaped.Flow cytometry results showed that CD90+/CD29+/CD45-cells were upto99%of P3BMSCs.During adipogenic induction, small lipid droplets accumulated andbecame larger gradually in some cells. The induced cells were positive for oilred O staining after2weeks. During osteogenic induction, BMSCs graduallytransformed to polygonal or irregular-shaped cells and grew intensively. Theinduced cells were positive for alizarin red staining after2weeks. Duringcartilage induction, the local part of BMSCs gradually changed into round oroval and grew rapidly and intensively to form nodular structures. After2weeks, the nodular structures were positive for alcian blue staining.2Exogenous genes of Nkx2.5, GATA4, TBX5induced BMSCs todifferentiate into myocardial cells.The experiment confirmed that the appropriate cell seeding density is4×104/cm2, the best transfection system is the plasmid DNA（μg） andLipofectamine2000（μl） at a ratio of1:2.5.Results of Western blot at48hours after transfection showed thatNkx2.5-EGFP fusion protein expressed in A1group, whereas none in groupA2or A3. The myc protein expressed in B1group, while none in group B2orB3. The TBX5protein expressed in group C1, C2and C3, but that in C1groupwas the highest （P<0.05）. The results showed that plasmid pEGFP-N1-Nkx2.5,pVP22-GATA-4/myc-His and pcDNA3.1-TBX5can transfect BMSCs bycationic liposome reagent.Four weeks after the exogenous gene transfection and the followingculture, some cells of group A1, B1and C1elongated and widened, and thelocal cells grew intensively. Few cells in the other groups changed similarly.Four weeks after the exogenous gene transfection and the followingculture, immunocytochemistry results showed that cTnT and Cx43expressedin group A1, B1and C1. There were brown filament-like or granular structuresin the cytoplasm of cTnT positive cells, and tan particles in the cytoplasm of Cx43ones. Few positive cells were in the other groups. The cTnT and Cx43expressed in group A1, A2and A3, but these in A1group were the highest（P<0.05）. The cTnT and Cx43expressed in group B1, B2and B3, but these inB1group were the highest （P<0.05）. The cTnT and Cx43expressed in groupC1, C2and C3, but these in C1group were the highest （P<0.05）.Four weeks after the exogenous gene transfection and the followingculture, Western blot results showed that cTnT expressions of transfectedgroups were significinatly higher than those of empty plasmid groups andblank ones. The results were identical with immunocytochemistry results.Real Time PCR results showed that Cx43expression level trends of A1,B1and C1group were similar. They increased gradually at1^stweek, reached apeak at2^ndor3^rdweek, followed by expression down. β-MHC expressionlevel of group A1from1^stto3^rdweek were significantly higher than that of P3BMSCs （P<0.05）, then decreased at4^thweek, without significant differencewith that of P3BMSCs （P>0.05）. β-MHC expression of group B1was highestat3rdweek （P<0.05）, then decreased. There were no significant differenceamong1^st,2^nd,4^thweek and P3BMSCs （P>0.05）. β-MHC expression of groupC1increased gradually and was highest at4^thweek （P<0.05）. There were nosignificant difference among1^st,2^nd, week and P3BMSCs （P>0.05）. MLC-2expression levels of group A1and C1at1^stweek had no significant differencewith that of P3BMSCs （P>0.05）, then significantly increased at2ndweek（P<0.05）, decreased gradually at3rdweek. The expression was weak at4^thweek, without significant difference with that of P3BMSCs （P>0.05）. MLC-2expression of group C1was highest at3rdweek （P<0.05）, then decreased.There were no significant difference among1^st,2^nd,4^thweek and P3BMSCs（P>0.05）.3Combination of exogenous genes of Nkx2.5, GATA4, TBX5and oxytocininduced BMSCs into myocardial cells.Four weeks after the exogenous gene transfection and the followingculture, some cells of all the groups except blank one elongated and widened,and the local cells grew intensively. Few cells in blank group changed similarly.Four weeks after the exogenous gene transfection and the followingculture, immunocytochemistry results showed that cTnT and Cx43expressedin all the groups except blank one. There were brown filament-like or granularstructures in the cytoplasm of cTnT positive cells, and tan particles in thecytoplasm of Cx43ones. Few positive cells were in blank group. The cTnTexpressions of transfection groups and OT group were high, oftransfection+induced drug groups were the highest, of blank group were little.There were significant difference between induced groups and blank group（P<0.05）. The Cx43expressions of transfection groups, transfection+induceddrug groups and OT group were high, of blank group were little. There weresignificant difference between induced groups and blank group （P<0.05）.Western blot results showed that the cTnT expressions of transfectiongroups and OT group were high, of transfection+induced drug groups werethe highest, of blank group were little. There were significant differencebetween induced groups and blank group （P<0.05）. The results were identicalwith immunocytochemistry results.Observation of the ultrastructure by transmission electron microscopyshowed P3BMSCs and blank group cells were spindle-shaped and riched withmany cell organelles such as ribosomes, mitochondria, rough endoplasmicreticulum, lysosomes in the cytoplast. Gap junctions were visible occasionallybetween a few of cells. The cells of transfection groups, transfection+OTgroups and OT group were spindle-shaped and riched with many cellorganelles such as mitochondria, ribosomes, rough endoplasmic reticulum,lysosomes in the cytoplast. The oval nucleus located in the cell central. Manyparalleled myofilament-like structures with dense zone appeared around thenucleus and more were in the side or end of the cytoplast. More gap junctionswere observed in the side or end of the cells. More myofilament-like structuresand gap junctions appeared in the cells of transfection groups andtransfection+OT groups than those in the cells of OT group. Conclusion:1SD rat BMSCs were isolated, amplified and characterized successfully.2The plasmids of pEGFP-N1-Nkx2.5, pVP22-GATA-4/myc-His andpcDNA3.1-TBX5transfected BMSCs by cationic liposome reagent,Lipofectamine2000.3SD rat BMSCs were transfected with the heart early transcription factorof Nkx2.5, GATA4and TBX5, and differentiated toward myocardial cells.The differentiated cells expressed myocardial specific markers, cTnT, Cx43,β-MHC and MLC-2, in both gene level and protein level, and had earlymyocardial cells’ structural characteristics.4OT induced SD rat BMSCs to differentiate toward myocardial cells.The differentiated cells had early myocardial cells’ structural characteristics.5There were superimposed effect of cardiomyocyte differentiationbetween Nkx2.5, GATA4or TBX5transfection and OT induction. Thecombination method gained the highest expression level of cTnT, but didn’t ofCx43.更多还原