外源性基因Nkx2.5、GATA-4转染兔BMSCs与心肌细胞共培养后治疗心肌梗死的实验研究
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摘要
目的：心血管疾病是目前世界上主要危害人类健康的疾患之一，其发病率很高。一旦发生急性心肌梗塞（acute myocardial infarction，AMI）或者其他的心脏病等，会造成成活心肌细胞数量下降，瘢痕组织形成，心室重构，心肌收缩能力下降，从而导致心功能下降，甚至最终诱发心力衰竭而危及生命。目前采用外源性的干细胞移植，将外源性的干细胞移植到梗死心肌周边，发挥其综合生物学作用，从而改善心脏功能，这是个极具发展潜力的治疗方法。最近几十年中，科学家们为寻找一种能够修复损伤心肌、恢复心脏功能的干细胞投入了极大的热情、努力和精力，目前研究最多的是骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs），与其他干细胞相比其具有明显的优势：①来源丰富广泛，容易获取，可以自体移植，不涉及伦理道德等问题。②容易培养分离，可快速增殖，其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的稳定性良好。③细胞本身具有多种、多向分化能力。④其本身具备低免疫原性和高可移植性。因此，骨髓间充质干细胞是心血管疾病干细胞移植治疗的较理想的一种种子细胞。
骨髓间充质干细胞体外心肌细胞分化的方法常见的有三种：①药物诱导；②与心肌细胞共培养；③基因修饰。然而骨髓间充质干细胞体外心肌细胞分化诱导的低效率是目前存在的主要问题，如何能够提高骨髓间充质干细胞体外心肌细胞诱导分化的效率成为科研人员研究的热点。
对骨髓间充质干细胞进行基因修饰，从分子水平促进骨髓间充质干细胞向心肌细胞分化，是近几年伴随分子生物学技术发展而新发展的诱导方法，旨在通过启动某个或某些关键基因，激活心肌分化基因的调控网络，实现骨髓间充质干细胞向心肌细胞的分化。心脏早期转录因子在心脏发育早期的表达，其中最主要的有Nkx2.5、GATA4、TBX5等，它们能调控许多心脏结构蛋白基因的表达，促进其向心肌细胞方向分化，对心脏正常发育具有重要的作用。而药物诱导向心肌细胞分化的文献报道有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。5-氮胞苷是目前最常用的诱导剂，但其存在一定的细胞药物毒性，对于临床治疗存在一定的风险。与心肌细胞共培养则是目前比较常见的一种骨髓间充质干细胞体外心肌细胞诱导分化方法，其体外培养也较容易，而且此方法细胞毒性几乎不存在，对于临床治疗有一定的可行性。
本实验采用心脏早期转录因子Nkx2.5、GATA4转染骨髓间充质干细胞后，再与心肌细胞体外共培养的方法，探讨Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境培养对骨髓间充质干细胞向心肌细胞分化的作用和影响，同时将Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境共培养诱导骨髓间充质干细胞注射入兔梗死模型的梗死心肌周边，观察其对梗死心肌的影响效果，为实现骨髓间充质干细胞高效的心肌细胞分化提供实验依据，并为干细胞移植修复心肌损伤提供良好的细胞来源和临床基础。
方法：
1新西兰兔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严格控制消化时间的传代方法结合，分离、纯化、扩增培养新西兰兔骨髓间充质干细胞。从三个方面对分离扩增培养的骨髓间充质干细胞进行鉴定：（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。
2外源基因Nkx2.5、GATA4单纯诱导兔BMSCs和其与心肌细胞外环境共培养诱导骨髓间充质干细胞向心肌细胞分化。
实验分组：A1组（单纯转染pEGFP-N1-Nkx2.5）、A2组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、A3组（骨髓间充质干细胞培养空白组）；B1组（单纯转染pVP22-GATA-4/myc-His）、B2组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）、B3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转染各组骨髓间充质干细胞。转染与共培养后48h经Western blot检测各组外源基因表达。而后继续培养4周，免疫细胞化学、Western blot检测各组细胞心肌特异性心肌肌钙蛋白T（cardiac troponinT，cTnT）、连接蛋白43（connexin43，Cx43）的表达。
3外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔骨髓间充质干细胞对兔心肌梗死模型修复梗死心肌的实验研究。
实验分组：采用结扎左冠状动脉前降支的方法建立兔心肌梗塞模型，每组选造模成功兔10只。实验分组：A组：转染pEGFP-N1-Nkx2.5与心肌细胞共培养BMSCs局部10只注射；B组：转染pVP22-GATA-4/myc-His与心肌细胞共培养BMSCs局部10只注射；C组：骨髓间充质干细胞培养空白组BMCs10只注射，其中分组的兔排除了在建模、细胞培养、心肌内注射等任一环节失败的实验动物。于细胞移植4周后取材，福尔马林固定，HE染色及免疫组织化学染色，光学纤维镜观察移植细胞的存活和分布。
结果：
1骨髓间充质干细胞的分离、培养和鉴定。
首先从兔长骨骨髓中分离原代骨髓间充质干细胞，48h后首次换液，可见贴壁细胞呈圆形、多角形、梭形，以梭形居多，细胞生长较慢。2～3天后贴壁细胞生长迅速，相邻细胞逐渐汇合聚团，4～5天后细胞汇合可达80%，呈旋涡状或放射状聚集生长。第1次传代后骨髓间充质干细胞贴壁比原代细胞快，细胞分布均匀。随着细胞换液和传代，贴壁的骨髓间充质干细胞形态逐渐趋于一致；传代至第3代（P3）时骨髓间充质干细胞形态基本单一，呈长梭形。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，说明获得的骨髓间充质干细胞具有较高的均一性，细胞纯度较高。
2外源基因Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境共培养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4×104/cm2密度传代，在次日转染时达到90%～95%汇合；质粒DNA和转染试剂Lipofectamine2000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、A2组有外源性Nkx2.5表达，而A3空白组无表达，A1组、A2组较A3空白组表达显著增高（P<0.05）；检测B1、B2、B3各组myc蛋白表达，结果表明转染组B1组、B2组有外源性GATA4表达，而B3空白组无表达，B1组、B2组较B3空白组表达显著增高（P<0.05）。
外源基因转染培养4周后形态学观察，A1组、B1组细胞呈长梭形，继续延长培养时间，可见局部细胞密集生长，当细胞密度达到一定程度后，细胞不再生长，A1组、B1组细胞形态改变相似；A2组、B2组细胞与心肌细胞聚集生长大部为梭形，部分细胞生长情况较好的区域可见细胞团的节奏性跳动。A3组、B3组细胞密集排列生长，呈长梭形，可见局部细胞密集重叠生长。
外源基因转染培养4周后，免疫细胞化学检测结果表明：A1组、B1组、A2组、B2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。A1组、B1组、A2组、B2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。A3组、B3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、A2组的cTnT、Cx43表达高，其中A2组的cTnT、Cx43表达最高，空白A3组cTnT、Cx43表达较低，与A1组、A2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、B2组的cTnT、Cx43表达高，其中B2组cTnT、Cx43表达最高，空白B3组cTnT、Cx43表达较低，与B1组、B2组相比均有显著差异（P<0.05）。
外源基因转染培养4周后，Western blot结果显示转染组与转染共培养组cTnT蛋白呈明显高表达，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。
3外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔骨髓间充质干细胞修复兔梗死心肌的效果。
局部移植的经过诱导培养的新西兰兔骨髓间充质干细胞可在新西兰兔梗死心肌存活、迁移分布，并能分化为心肌样细胞。
4周后，行HE染色，A组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、B组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）心肌细胞间有移植细胞存活生长，细胞在移植中央区呈团状生长，并在移植周边区放射状浸润生长，移植区与正常心肌交界处的移植细胞逐渐移形为梭形。可见成纤维细胞和新生血管，部分新西兰白兔骨髓间充质干细胞与心肌细胞之间可见有新生细胞连接生成。C（骨髓间充质干细胞培养空白组）少见移植细胞心肌细胞分化和新生血管形成。A1组、B组的观察实验效果优于C组。行免疫组织化学染色，A组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、B组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）移植后，心肌细胞间有移植细胞存活生长，部分细胞周边可见棕色染色物质，说明移植细胞已能成功存活于心肌细胞之间，或是部分移植细胞已能够被诱导分化为心肌样细胞，可以增加心肌细胞间连接，促进受损的心肌组织进行修复，而部分带有棕色物质的细胞已经能自我聚集，形成血管样物质，这在一定程度增加了受损心肌内的血液循环，也能促进受损的心肌组织进行修复，在移植细胞区域，可见移植细胞呈团状生长，并在移植周边区放射状浸润生长，移植区与正常心肌交界处的移植细胞逐渐移形为梭形，两者之间的界限模糊，均可见到棕色物质，说明移植细胞已能部分或完全替代受损心肌细胞的部分功能。部分移植细胞与心肌细胞之间可见有新生细胞连接生成。C组（骨髓间充质干细胞培养空白组）则少见移植细胞存活及棕色染色细胞。说明经诱导后再与心肌细胞共培养的新西兰白兔骨髓间充质干细胞在移植后的心肌内微环境的适应能力上更好，能更为快速的发挥移植细胞自身的功能，同时也能提高移植细胞的存活率。
结论：
1成功的对兔骨髓间充质干细胞进行分离、培养扩增及鉴定。
2成功进行外源基因Nkx2.5、GATA4转染兔骨髓间充质干细胞及鉴定，外源基因Nkx2.5、GATA4转染和其与心肌细胞外环境共培养诱导骨髓间充质干细胞向心肌细胞分化的效果优于单纯依靠外源基因Nkx2.5、GATA4诱导骨髓间充质干细胞向心肌细胞分化。
3成功完成新西兰兔心肌梗死模型的制作，外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔BMSCs能够在新西兰白兔的梗死心肌存活、迁移分布，并能分化为心肌样细胞，部分修复、缓解、活化兔的梗死心肌，效果优于单纯兔骨髓间充质干细胞。
Objective: Cardiovascular disease is one of the world's major hazard tohuman health disorders, the incidence rate is very high. In the event of acutemyocardial infarction (acute myocardial infarction, AMI) or heart disease, cancause survived myocardial cells decline, scar tissue formation, ventricularreconstruction, decreased myocardial contractility, resulting in decreased heartfunction, and even eventually induce heart failure and life-threatening.Exogenous cell transplantation, exogenous cells transplanted into infarctedmyocardium surrounding plays its biological effects, thereby improvingcardiac function, which is a potential treatment. Recent decades, scientistslooking for a way to repair the injured myocardium and restore cardiacfunction in stem cells has invested a great deal of enthusiasm, effort andenergy, the most studied bone marrow mesenchymal stem cells (bone marrowmesenchymal Stem Cells BMSCs), compared with other stem cells which hasobvious advantages:(1) a rich source of a wide range of easily accessible,autologous transplantation, does not involve ethical issues.②easy separationof culture, the proliferation of fast, highly cultured in vitro amplificationability, and good genetic stability after multiple passages in vitro.(3) cell witha variety of multi-differentiation capacity.④having low immunogenicity andhigh portability. Therefore, BMSCs cardiovascular diseases stem celltransplantation in the treatment of an ideal seed cells.
Bone marrow mesenchymal stem cells in vitro cardiac myocytedifferentiation. There are three common:(1) drug-induced;(2) and myocardialcell co-culture;(3) genetically modified. Bone marrow mesenchymal stemcells in vitro myocardial cell differentiation induced inefficiencies, however, iscurrently the main problems, how can we improve the efficiency of bone marrow mesenchymal stem cells induced to differentiate in vitro cardiacmyocyte research focus of the researchers.
Genetically modified bone marrow mesenchymal stem cells from themolecular level to promote the differentiation of bone marrow mesenchymalstem cells into cardiomyocytes, in recent years, accompanied by thedevelopment of molecular biology techniques and new developments inducedthe purpose by starting athe key gene activation myocardial differentiationgene regulatory networks, bone marrow mesenchymal stem cell differentiationinto cardiomyocytes. Early expression of early cardiac transcription factors incardiac development, the most important Nkx2.5, GATA4, TBX5, they canregulate gene expression of cardiac structural proteins, promoting theirdifferentiation into cardiomyocytes direction, the normal development of theheart has role.5-Azacytidine (5-azacytidine,5-aza), DMSO(Dimethylsulfoxide, DMSO), insulin, dexamethasone, ascorbic acid and otherdrug-induced myocardial cells reported in the literature.5-azacytidine is themost commonly used inducer, but there is a certain cell toxicity, there is acertain risk for clinical treatment. Myocardial cell co-culture is a relativelycommon form of bone marrow mesenchymal stem cells in vitro myocardialcells induced to differentiate methods, in vitro culture is easier, and thecytotoxicity of this method is almost non-existent, is feasible for clinicaltreatment.
In this study, early cardiac transcription factor Nkx2.5of GATA4transfected bone marrow mesenchymal stem cells, and cardiac myocytes invitro co-culture, explore Nkx2.5of GATA4simple induction and with the themyocardial extracellular environment culture of bone marrowthe role andimpact of the differentiation of mesenchymal stem cells into cardiomyocytes,to provide experimental evidence for myocardial cell differentiation of bonemarrow mesenchymal stem cells efficient, good source of cells for stem cellsfor repair of myocardial injury and clinical basis.
Methods:
1A bone marrow mesenchymal stem cell isolation, culture and identification.
Adherent separation screening method was changed, combined with strictcontrol of the passage of the digestion time, separation, purification, werecultured rabbit bone marrow mesenchymal stem cells. Separations werecultured bone marrow mesenchymal stem cells were identified from threeaspects:(1) observed the adhesion growth characteristics of cultured cells;(2)by flow cytometry combined detection of the expression of cell surfacemolecules CD90, CD29, CD45.
2Exogenous gene Nkx2.5, GATA4simple induction and induced bonemarrow mesenchymal stem cells differentiate into myocardial cellsco-cultured with myocardial extracellular environment.
Experimental groups: group A1(simple transfer dye pEGFP-N1-Nkx2.5),A2group (transfected dye pEGFP-N1-Nkx2.5and myocardial cell co-culture),A3group (bone marrow mesenchymal stem cells blank group);B1group (onlythe transfection pVP22-GATA-4/myc-His), B2group (transfectedpVP22-GATA-4/myc-His myocardial cell co-culture), B3group (blank controlgroup); prior to transfection by the pre-experiments to determine optimal celldensity transfection, the the best transfection system, using cationictransfection reagent Lipofectamine2000transfection group of BMSCs.48hafter transfection and common culture by Western blot to detect the expressionof foreign genes. Then continue to foster4weeks, immunocytochemistry,Western blot detection of cells in each group of cardiac troponin T (cardiactroponin T, cTnT), connexin43(connexin43, Cx43) expression.
3Exogenous gene Nkx2.5co-culture the Gata4induced myocardialextracellular environment of New Zealand rabbit bone marrow mesenchymalstem cells to repair rabbit myocardial infarction results.
Experimental groups: A group (transfected with pEGFP-N1-Nkx2.5andmyocardial cells co-cultured)10injection; B group (transfectedpVP22-GATA-4/myc-His myocardial cell co-culture)10injection; C group(bone marrow mesenchymalstem cell culture control group)10injection.Ligation of the left anterior descending branch established rabbit model of myocardial infarction. Where in grouping rabbits excluded a failure in any onepart of the modeling, cell culture, intramyocardial injection of experimentalanimals. In stem cells4weeks after transplantation, the survival anddistribution of transplanted cells by HE staining.
Results:
1Bone marrow mesenchymal stem cells, culture and identification.
Separation of primary bone marrow-derived mesenchymal stem cellsafter48h the medium was changed for the first time, showing adherent cellswere round, polygonal, spindle, spindle mostly slow cell growth.2to3daysfor adherent cells rapidly the adjacent cells gradually merging agglomerate,4to5days after confluence up to80%, showed a spiral-shaped or radialaggregation growth.1st passage adherent bone marrow mesenchymal stemcells faster than primary cells, the cells evenly distributed. As the cell mediumwas changed and passaged adherent bone marrow mesenchymal stem cellmorphology is becoming more and more consistent; passage to the3rdgeneration of bone marrow mesenchymal stem cells form the basic singleelongated spindle. Flow cytometry Growth good P3cells in the CD90+/CD29+/CD45-cells than99%, to obtain bone marrow mesenchymal stemcells have higher homogeneity and higher cell purity.
2Exogenous gene Nkx2.5, GATA4simple induction and its co-culturedwith myocardial extracellular environment induces differentiation of bonemarrow mesenchymal stem cells into cardiomyocytes.
Found by preliminary experiments, in the next day of transfection:passaged at a density of4×104/cm2reaches90%~95%confluence; plasmidDNA (ug) and the transfection reagent Lipofectamine2000(microl)'s, aratio of1:2.5,this condition transfection achieved good results.
48h after transfection, Western blot detection of A1, A2, A3groupsNkx2.5: EGFP fusion protein expression, the results showed that transfectiongroup A1, A2group of exogenous Nkx2.5expression, while the A3blankgroup expression.group A1, group A2, A3blank group was significantlyhigher (P <0.05); detection B1, B2, B3of myc protein expression, the results showed that transfection group group B1, B2group the expression ofexogenous GATA4, and B3is blankThere was no expression, B1group, groupB2, B3blank group was significantly higher (P <0.05).
Exogenous gene transfection and incubated for4weeks aftermorphological observation group A1, B1cells fusiform, some cells increases,widened further extended incubation time, showing that the local cell densitygrowth, when the cell density reaches a certain level, the cells no longer thegrowth of cells similar to the morphological changes; group A2, B2cells andcardiac myocytes aggregation growth in most spindle cell growth better areavisible rhythmic beating of the cell mass. Group A3, B3cells arranged indense growth, fusiform visible local cell density overlap growth.
Exogenous gene into cultured4weeks, immunocytochemistry resultsshow that: group A1, B1group, group A2, B2group some cells positive forcTnT, cTnT positive cells cytoplasm visible brownish yellow color silk meshand particle-like structure. A1, B1, A2, B2group of cells were Cx43positive,visible brown granules in the cytoplasm of Cx43-positive cells. Group A3, B3group, only a small number of cells cTnT positive Cx43expression. Eachgroup integrated absorbance (IA) value of statistical results: groups A1, A2Group A3group, transfection group group A1, A2group cTnT, Cx43expression, in which the group A2cTnT, Cx43expression, blank A3groupcTnT, Cx43expression is low, group A1, A2group compared were significantdifferences (P <0.05); B1group, group B2, B3group, transfection groupgroup B1, B2group cTnT Cx43expression group B2cTnT, Cx43expression,blank B3group cTnT, Cx43expression is low, group B1, B2group weresignificantly different (P <0.05).
Exogenous gene transfection after4weeks of culture, the Western blotresults showed that transfection group and transfected co-culture group cTnTprotein was highly expressed in the blank group almost no expression.Statistical results consistent immunocytochemistry cTnT test results.
3. Exogenous gene Nkx2.5co-culture the Gata4induced myocardialextracellular environment of New Zealand rabbit bone marrow mesenchymal stem cells to repair rabbit myocardial infarction results.
Bone marrow mesenchymal stem cells in the infarcted rabbit heartsurvival, migration and distribution, and can differentiate intocardiomyocyte-like cells.
Four weeks after HE staining, myocardial cells there is the growth of thetransplanted cells survive, the cells in the transplant central zone Movementslug growth, and in the transplantation the peripheral zone Radiologists shapedinfiltrative growth, transplant area and normal myocardium at the junction atthe transplanted cells gradually shift shaped as spindle. Visible fibroblasts andneovascularization. New Zealand rabbit bone marrow mesenchymal stem cellsseen around the new cells.
Conclusion:
1Successfully isolated rabbit bone marrow mesenchymal stem cells,cultured amplification and identification.
2Successful exogenous gene Nkx2.5, GATA4transfection of rabbit bonemarrow mesenchymal stem cells and identification. Exogenous gene Nkx2.5,Gata4turn stained and myocardial extracellular environmentco-culture-induced differentiation of bone marrow mesenchymal stem cellsinto cardiomyocytes is better than relying solely on foreign gene Nkx2.5,GATA4-induced bone marrow mesenchymal stem cells intodifferentiation ofcardiomyocytes.
3Successful completion of the New Zealand rabbit model of myocardialinfarction production. Exogenous gene Nkx2.5, co-culture the Gata4inducedmyocardial extracellular environment of New Zealand rabbit bone marrowmesenchymal stem cells can repair, mitigation, activation of rabbit myocardialinfarction.
骨髓间充质干细胞体外心肌细胞分化的方法常见的有三种：①药物诱导；②与心肌细胞共培养；③基因修饰。然而骨髓间充质干细胞体外心肌细胞分化诱导的低效率是目前存在的主要问题，如何能够提高骨髓间充质干细胞体外心肌细胞诱导分化的效率成为科研人员研究的热点。
对骨髓间充质干细胞进行基因修饰，从分子水平促进骨髓间充质干细胞向心肌细胞分化，是近几年伴随分子生物学技术发展而新发展的诱导方法，旨在通过启动某个或某些关键基因，激活心肌分化基因的调控网络，实现骨髓间充质干细胞向心肌细胞的分化。心脏早期转录因子在心脏发育早期的表达，其中最主要的有Nkx2.5、GATA4、TBX5等，它们能调控许多心脏结构蛋白基因的表达，促进其向心肌细胞方向分化，对心脏正常发育具有重要的作用。而药物诱导向心肌细胞分化的文献报道有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。5-氮胞苷是目前最常用的诱导剂，但其存在一定的细胞药物毒性，对于临床治疗存在一定的风险。与心肌细胞共培养则是目前比较常见的一种骨髓间充质干细胞体外心肌细胞诱导分化方法，其体外培养也较容易，而且此方法细胞毒性几乎不存在，对于临床治疗有一定的可行性。
本实验采用心脏早期转录因子Nkx2.5、GATA4转染骨髓间充质干细胞后，再与心肌细胞体外共培养的方法，探讨Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境培养对骨髓间充质干细胞向心肌细胞分化的作用和影响，同时将Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境共培养诱导骨髓间充质干细胞注射入兔梗死模型的梗死心肌周边，观察其对梗死心肌的影响效果，为实现骨髓间充质干细胞高效的心肌细胞分化提供实验依据，并为干细胞移植修复心肌损伤提供良好的细胞来源和临床基础。
方法：
1新西兰兔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严格控制消化时间的传代方法结合，分离、纯化、扩增培养新西兰兔骨髓间充质干细胞。从三个方面对分离扩增培养的骨髓间充质干细胞进行鉴定：（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。
2外源基因Nkx2.5、GATA4单纯诱导兔BMSCs和其与心肌细胞外环境共培养诱导骨髓间充质干细胞向心肌细胞分化。
实验分组：A1组（单纯转染pEGFP-N1-Nkx2.5）、A2组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、A3组（骨髓间充质干细胞培养空白组）；B1组（单纯转染pVP22-GATA-4/myc-His）、B2组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）、B3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转染各组骨髓间充质干细胞。转染与共培养后48h经Western blot检测各组外源基因表达。而后继续培养4周，免疫细胞化学、Western blot检测各组细胞心肌特异性心肌肌钙蛋白T（cardiac troponinT，cTnT）、连接蛋白43（connexin43，Cx43）的表达。
3外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔骨髓间充质干细胞对兔心肌梗死模型修复梗死心肌的实验研究。
实验分组：采用结扎左冠状动脉前降支的方法建立兔心肌梗塞模型，每组选造模成功兔10只。实验分组：A组：转染pEGFP-N1-Nkx2.5与心肌细胞共培养BMSCs局部10只注射；B组：转染pVP22-GATA-4/myc-His与心肌细胞共培养BMSCs局部10只注射；C组：骨髓间充质干细胞培养空白组BMCs10只注射，其中分组的兔排除了在建模、细胞培养、心肌内注射等任一环节失败的实验动物。于细胞移植4周后取材，福尔马林固定，HE染色及免疫组织化学染色，光学纤维镜观察移植细胞的存活和分布。
结果：
1骨髓间充质干细胞的分离、培养和鉴定。
首先从兔长骨骨髓中分离原代骨髓间充质干细胞，48h后首次换液，可见贴壁细胞呈圆形、多角形、梭形，以梭形居多，细胞生长较慢。2～3天后贴壁细胞生长迅速，相邻细胞逐渐汇合聚团，4～5天后细胞汇合可达80%，呈旋涡状或放射状聚集生长。第1次传代后骨髓间充质干细胞贴壁比原代细胞快，细胞分布均匀。随着细胞换液和传代，贴壁的骨髓间充质干细胞形态逐渐趋于一致；传代至第3代（P3）时骨髓间充质干细胞形态基本单一，呈长梭形。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，说明获得的骨髓间充质干细胞具有较高的均一性，细胞纯度较高。
2外源基因Nkx2.5、GATA4单纯诱导和其与心肌细胞外环境共培养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4×104/cm2密度传代，在次日转染时达到90%～95%汇合；质粒DNA和转染试剂Lipofectamine2000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、A2组有外源性Nkx2.5表达，而A3空白组无表达，A1组、A2组较A3空白组表达显著增高（P<0.05）；检测B1、B2、B3各组myc蛋白表达，结果表明转染组B1组、B2组有外源性GATA4表达，而B3空白组无表达，B1组、B2组较B3空白组表达显著增高（P<0.05）。
外源基因转染培养4周后形态学观察，A1组、B1组细胞呈长梭形，继续延长培养时间，可见局部细胞密集生长，当细胞密度达到一定程度后，细胞不再生长，A1组、B1组细胞形态改变相似；A2组、B2组细胞与心肌细胞聚集生长大部为梭形，部分细胞生长情况较好的区域可见细胞团的节奏性跳动。A3组、B3组细胞密集排列生长，呈长梭形，可见局部细胞密集重叠生长。
外源基因转染培养4周后，免疫细胞化学检测结果表明：A1组、B1组、A2组、B2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。A1组、B1组、A2组、B2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。A3组、B3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、A2组的cTnT、Cx43表达高，其中A2组的cTnT、Cx43表达最高，空白A3组cTnT、Cx43表达较低，与A1组、A2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、B2组的cTnT、Cx43表达高，其中B2组cTnT、Cx43表达最高，空白B3组cTnT、Cx43表达较低，与B1组、B2组相比均有显著差异（P<0.05）。
外源基因转染培养4周后，Western blot结果显示转染组与转染共培养组cTnT蛋白呈明显高表达，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。
3外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔骨髓间充质干细胞修复兔梗死心肌的效果。
局部移植的经过诱导培养的新西兰兔骨髓间充质干细胞可在新西兰兔梗死心肌存活、迁移分布，并能分化为心肌样细胞。
4周后，行HE染色，A组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、B组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）心肌细胞间有移植细胞存活生长，细胞在移植中央区呈团状生长，并在移植周边区放射状浸润生长，移植区与正常心肌交界处的移植细胞逐渐移形为梭形。可见成纤维细胞和新生血管，部分新西兰白兔骨髓间充质干细胞与心肌细胞之间可见有新生细胞连接生成。C（骨髓间充质干细胞培养空白组）少见移植细胞心肌细胞分化和新生血管形成。A1组、B组的观察实验效果优于C组。行免疫组织化学染色，A组（转染pEGFP-N1-Nkx2.5与心肌细胞共培养）、B组（转染pVP22-GATA-4/myc-His与心肌细胞共培养）移植后，心肌细胞间有移植细胞存活生长，部分细胞周边可见棕色染色物质，说明移植细胞已能成功存活于心肌细胞之间，或是部分移植细胞已能够被诱导分化为心肌样细胞，可以增加心肌细胞间连接，促进受损的心肌组织进行修复，而部分带有棕色物质的细胞已经能自我聚集，形成血管样物质，这在一定程度增加了受损心肌内的血液循环，也能促进受损的心肌组织进行修复，在移植细胞区域，可见移植细胞呈团状生长，并在移植周边区放射状浸润生长，移植区与正常心肌交界处的移植细胞逐渐移形为梭形，两者之间的界限模糊，均可见到棕色物质，说明移植细胞已能部分或完全替代受损心肌细胞的部分功能。部分移植细胞与心肌细胞之间可见有新生细胞连接生成。C组（骨髓间充质干细胞培养空白组）则少见移植细胞存活及棕色染色细胞。说明经诱导后再与心肌细胞共培养的新西兰白兔骨髓间充质干细胞在移植后的心肌内微环境的适应能力上更好，能更为快速的发挥移植细胞自身的功能，同时也能提高移植细胞的存活率。
结论：
1成功的对兔骨髓间充质干细胞进行分离、培养扩增及鉴定。
2成功进行外源基因Nkx2.5、GATA4转染兔骨髓间充质干细胞及鉴定，外源基因Nkx2.5、GATA4转染和其与心肌细胞外环境共培养诱导骨髓间充质干细胞向心肌细胞分化的效果优于单纯依靠外源基因Nkx2.5、GATA4诱导骨髓间充质干细胞向心肌细胞分化。
3成功完成新西兰兔心肌梗死模型的制作，外源基因Nkx2.5、GATA4诱导与心肌细胞外环境共培养后的新西兰兔BMSCs能够在新西兰白兔的梗死心肌存活、迁移分布，并能分化为心肌样细胞，部分修复、缓解、活化兔的梗死心肌，效果优于单纯兔骨髓间充质干细胞。
Objective: Cardiovascular disease is one of the world's major hazard tohuman health disorders, the incidence rate is very high. In the event of acutemyocardial infarction (acute myocardial infarction, AMI) or heart disease, cancause survived myocardial cells decline, scar tissue formation, ventricularreconstruction, decreased myocardial contractility, resulting in decreased heartfunction, and even eventually induce heart failure and life-threatening.Exogenous cell transplantation, exogenous cells transplanted into infarctedmyocardium surrounding plays its biological effects, thereby improvingcardiac function, which is a potential treatment. Recent decades, scientistslooking for a way to repair the injured myocardium and restore cardiacfunction in stem cells has invested a great deal of enthusiasm, effort andenergy, the most studied bone marrow mesenchymal stem cells (bone marrowmesenchymal Stem Cells BMSCs), compared with other stem cells which hasobvious advantages:(1) a rich source of a wide range of easily accessible,autologous transplantation, does not involve ethical issues.②easy separationof culture, the proliferation of fast, highly cultured in vitro amplificationability, and good genetic stability after multiple passages in vitro.(3) cell witha variety of multi-differentiation capacity.④having low immunogenicity andhigh portability. Therefore, BMSCs cardiovascular diseases stem celltransplantation in the treatment of an ideal seed cells.
Bone marrow mesenchymal stem cells in vitro cardiac myocytedifferentiation. There are three common:(1) drug-induced;(2) and myocardialcell co-culture;(3) genetically modified. Bone marrow mesenchymal stemcells in vitro myocardial cell differentiation induced inefficiencies, however, iscurrently the main problems, how can we improve the efficiency of bone marrow mesenchymal stem cells induced to differentiate in vitro cardiacmyocyte research focus of the researchers.
Genetically modified bone marrow mesenchymal stem cells from themolecular level to promote the differentiation of bone marrow mesenchymalstem cells into cardiomyocytes, in recent years, accompanied by thedevelopment of molecular biology techniques and new developments inducedthe purpose by starting athe key gene activation myocardial differentiationgene regulatory networks, bone marrow mesenchymal stem cell differentiationinto cardiomyocytes. Early expression of early cardiac transcription factors incardiac development, the most important Nkx2.5, GATA4, TBX5, they canregulate gene expression of cardiac structural proteins, promoting theirdifferentiation into cardiomyocytes direction, the normal development of theheart has role.5-Azacytidine (5-azacytidine,5-aza), DMSO(Dimethylsulfoxide, DMSO), insulin, dexamethasone, ascorbic acid and otherdrug-induced myocardial cells reported in the literature.5-azacytidine is themost commonly used inducer, but there is a certain cell toxicity, there is acertain risk for clinical treatment. Myocardial cell co-culture is a relativelycommon form of bone marrow mesenchymal stem cells in vitro myocardialcells induced to differentiate methods, in vitro culture is easier, and thecytotoxicity of this method is almost non-existent, is feasible for clinicaltreatment.
In this study, early cardiac transcription factor Nkx2.5of GATA4transfected bone marrow mesenchymal stem cells, and cardiac myocytes invitro co-culture, explore Nkx2.5of GATA4simple induction and with the themyocardial extracellular environment culture of bone marrowthe role andimpact of the differentiation of mesenchymal stem cells into cardiomyocytes,to provide experimental evidence for myocardial cell differentiation of bonemarrow mesenchymal stem cells efficient, good source of cells for stem cellsfor repair of myocardial injury and clinical basis.
Methods:
1A bone marrow mesenchymal stem cell isolation, culture and identification.
Adherent separation screening method was changed, combined with strictcontrol of the passage of the digestion time, separation, purification, werecultured rabbit bone marrow mesenchymal stem cells. Separations werecultured bone marrow mesenchymal stem cells were identified from threeaspects:(1) observed the adhesion growth characteristics of cultured cells;(2)by flow cytometry combined detection of the expression of cell surfacemolecules CD90, CD29, CD45.
2Exogenous gene Nkx2.5, GATA4simple induction and induced bonemarrow mesenchymal stem cells differentiate into myocardial cellsco-cultured with myocardial extracellular environment.
Experimental groups: group A1(simple transfer dye pEGFP-N1-Nkx2.5),A2group (transfected dye pEGFP-N1-Nkx2.5and myocardial cell co-culture),A3group (bone marrow mesenchymal stem cells blank group);B1group (onlythe transfection pVP22-GATA-4/myc-His), B2group (transfectedpVP22-GATA-4/myc-His myocardial cell co-culture), B3group (blank controlgroup); prior to transfection by the pre-experiments to determine optimal celldensity transfection, the the best transfection system, using cationictransfection reagent Lipofectamine2000transfection group of BMSCs.48hafter transfection and common culture by Western blot to detect the expressionof foreign genes. Then continue to foster4weeks, immunocytochemistry,Western blot detection of cells in each group of cardiac troponin T (cardiactroponin T, cTnT), connexin43(connexin43, Cx43) expression.
3Exogenous gene Nkx2.5co-culture the Gata4induced myocardialextracellular environment of New Zealand rabbit bone marrow mesenchymalstem cells to repair rabbit myocardial infarction results.
Experimental groups: A group (transfected with pEGFP-N1-Nkx2.5andmyocardial cells co-cultured)10injection; B group (transfectedpVP22-GATA-4/myc-His myocardial cell co-culture)10injection; C group(bone marrow mesenchymalstem cell culture control group)10injection.Ligation of the left anterior descending branch established rabbit model of myocardial infarction. Where in grouping rabbits excluded a failure in any onepart of the modeling, cell culture, intramyocardial injection of experimentalanimals. In stem cells4weeks after transplantation, the survival anddistribution of transplanted cells by HE staining.
Results:
1Bone marrow mesenchymal stem cells, culture and identification.
Separation of primary bone marrow-derived mesenchymal stem cellsafter48h the medium was changed for the first time, showing adherent cellswere round, polygonal, spindle, spindle mostly slow cell growth.2to3daysfor adherent cells rapidly the adjacent cells gradually merging agglomerate,4to5days after confluence up to80%, showed a spiral-shaped or radialaggregation growth.1st passage adherent bone marrow mesenchymal stemcells faster than primary cells, the cells evenly distributed. As the cell mediumwas changed and passaged adherent bone marrow mesenchymal stem cellmorphology is becoming more and more consistent; passage to the3rdgeneration of bone marrow mesenchymal stem cells form the basic singleelongated spindle. Flow cytometry Growth good P3cells in the CD90+/CD29+/CD45-cells than99%, to obtain bone marrow mesenchymal stemcells have higher homogeneity and higher cell purity.
2Exogenous gene Nkx2.5, GATA4simple induction and its co-culturedwith myocardial extracellular environment induces differentiation of bonemarrow mesenchymal stem cells into cardiomyocytes.
Found by preliminary experiments, in the next day of transfection:passaged at a density of4×104/cm2reaches90%~95%confluence; plasmidDNA (ug) and the transfection reagent Lipofectamine2000(microl)'s, aratio of1:2.5,this condition transfection achieved good results.
48h after transfection, Western blot detection of A1, A2, A3groupsNkx2.5: EGFP fusion protein expression, the results showed that transfectiongroup A1, A2group of exogenous Nkx2.5expression, while the A3blankgroup expression.group A1, group A2, A3blank group was significantlyhigher (P <0.05); detection B1, B2, B3of myc protein expression, the results showed that transfection group group B1, B2group the expression ofexogenous GATA4, and B3is blankThere was no expression, B1group, groupB2, B3blank group was significantly higher (P <0.05).
Exogenous gene transfection and incubated for4weeks aftermorphological observation group A1, B1cells fusiform, some cells increases,widened further extended incubation time, showing that the local cell densitygrowth, when the cell density reaches a certain level, the cells no longer thegrowth of cells similar to the morphological changes; group A2, B2cells andcardiac myocytes aggregation growth in most spindle cell growth better areavisible rhythmic beating of the cell mass. Group A3, B3cells arranged indense growth, fusiform visible local cell density overlap growth.
Exogenous gene into cultured4weeks, immunocytochemistry resultsshow that: group A1, B1group, group A2, B2group some cells positive forcTnT, cTnT positive cells cytoplasm visible brownish yellow color silk meshand particle-like structure. A1, B1, A2, B2group of cells were Cx43positive,visible brown granules in the cytoplasm of Cx43-positive cells. Group A3, B3group, only a small number of cells cTnT positive Cx43expression. Eachgroup integrated absorbance (IA) value of statistical results: groups A1, A2Group A3group, transfection group group A1, A2group cTnT, Cx43expression, in which the group A2cTnT, Cx43expression, blank A3groupcTnT, Cx43expression is low, group A1, A2group compared were significantdifferences (P <0.05); B1group, group B2, B3group, transfection groupgroup B1, B2group cTnT Cx43expression group B2cTnT, Cx43expression,blank B3group cTnT, Cx43expression is low, group B1, B2group weresignificantly different (P <0.05).
Exogenous gene transfection after4weeks of culture, the Western blotresults showed that transfection group and transfected co-culture group cTnTprotein was highly expressed in the blank group almost no expression.Statistical results consistent immunocytochemistry cTnT test results.
3. Exogenous gene Nkx2.5co-culture the Gata4induced myocardialextracellular environment of New Zealand rabbit bone marrow mesenchymal stem cells to repair rabbit myocardial infarction results.
Bone marrow mesenchymal stem cells in the infarcted rabbit heartsurvival, migration and distribution, and can differentiate intocardiomyocyte-like cells.
Four weeks after HE staining, myocardial cells there is the growth of thetransplanted cells survive, the cells in the transplant central zone Movementslug growth, and in the transplantation the peripheral zone Radiologists shapedinfiltrative growth, transplant area and normal myocardium at the junction atthe transplanted cells gradually shift shaped as spindle. Visible fibroblasts andneovascularization. New Zealand rabbit bone marrow mesenchymal stem cellsseen around the new cells.
Conclusion:
1Successfully isolated rabbit bone marrow mesenchymal stem cells,cultured amplification and identification.
2Successful exogenous gene Nkx2.5, GATA4transfection of rabbit bonemarrow mesenchymal stem cells and identification. Exogenous gene Nkx2.5,Gata4turn stained and myocardial extracellular environmentco-culture-induced differentiation of bone marrow mesenchymal stem cellsinto cardiomyocytes is better than relying solely on foreign gene Nkx2.5,GATA4-induced bone marrow mesenchymal stem cells intodifferentiation ofcardiomyocytes.
3Successful completion of the New Zealand rabbit model of myocardialinfarction production. Exogenous gene Nkx2.5, co-culture the Gata4inducedmyocardial extracellular environment of New Zealand rabbit bone marrowmesenchymal stem cells can repair, mitigation, activation of rabbit myocardialinfarction.
引文
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