| Objectives: Ischemic heart disease (IHD) is one of the most harmfuldiseases which severely threaten human life. Ischemia leads to a a fall innumbers of myocardial cells which have completely systolic and diastolicfunction. myocardial cells are terminally differentiated cells and can notregenerate after injury. The damaged cardiomyocytes only can be taken theplace of scar tissue,and the undamaged cardiomyocytes undergo hypertrophyin order to compensate part compensatory the loss of cardiac function. Finally,congestive heart failure would happened. Therefore, to increase the number ofcardiomyocytes which have completely systolic and diastolic function is thekey to cure the IHD. Currently,there are several methods to cure the IHD,suchas drug therapy, interventional, surgical and so on.Although these methodsdelayed the development of disease in some degree, they can not repair thedamaged cardiomyocytes and stop the progress of heart failure. Stem celltransplantation provides a new method for the reconstruction and functionrecovery of heart failure. Although at present is still in the basic and clinicalresearch stage, it brings new hope for patients with IHD. The results of thestudy show that stem cell transplantation can promote cardiovascular andmyocardial cells regeneration, improve myocardial blood supply, enhanceheart function.In many studies of cell transplantation, bone marrow mesenchymal stemcells cause widespread concern by the scientists for a broader clinicalapplication prospect. Bone marrow mesenchymal stem cells (bone marrowmesenchymal stem cells, BMSCs)，also called source of bone marrowstromal stem cells, are a subset of nonhematopoietic stem cells which hasmulti-differentiating potential and strong proliferative ability within the bone marrow. Under certain induction conditions, BMSCs can differentiate intomany non hematopoietic tissues, especially tissue cells rooting frommesoderm and neural ectoderm,Such as osteoblast, chondroblast, adipocyte,tendon cells, myocyte, neurocyte, etc. Basic and clinical studies haveconfirmed that BMSCs transplantation can repair the damaged myocardium,and improve cardiac function after myocardial infarction. Bone marrowmesenchymal stem cells which are easily available have high proliferation andmulti-lineage differentiation potential. It is convenient to import foreign geneinto BMSCs. After cell transplantation, they can still survive without immunerejection in the ischemic tissue. BMSCs become to be the main stem cells fortransplantation because of these advantages.GATA-4is a member of the GATA family of zinc finger transcriptionfactors, which widely expresses in tissue cells rooting from mesoderm andneural ectoderm.It plays important roles during development of the heart andis a significant symbols of the earliest precursor cells. GATA-4factor firstexpresses in the mesoderm of the heart, then in the endocardial dndmyocardium. The lack or mutations of the GATA-4will cause myocardialhypoplasia and endocardial cushion defect(TECD). In addition, GATA-4caninhibit myocardial apoptosis and adjust the survival of heart. In recent years,studies have confirmed that GATA-4also play an important role of regulationin the myocardial infarction repairment.Because it not only promoteangiogenesis, but also increase myocardial cell survival and differentiation.In this study, GATA-4was transfected into SD rat bone marrowmesenchymal stem cells by liposome.Then we observed the celldifferentiation in the myocardial cell cracking fluid,which was to mimicphysiological microenvironment, Compareing with the cells withouttransfection or microenvironment induction.and pure gene transfection ofbone marrow mesenchymal stem cells contrast to try to clarify the myocardialcell differentiation, some rules for the future effectively induced myocardialcell to provide strategy.We want to find the regulation during thedifferentiation of cardiomyocytes in order to provide strategy for the transplantation therapy.Methods:1The separation, purification and amplification of the SD rat’s bone marrowmesenchymal stem cellsBMSCs were isolated with the adherent culture method.Then they werecultivated in the DMEM/F12culture medium containing15%fetal bovineserum.We changed the solution on the third day for the first time,then in ainterval of3-4days. Passaged when the BMSCs covered80%~90%of thebottom. Take the third generation cells do the follow-up tests.2The culture of the SD rat’s myocardial cells dnd the preparation of themyocardial cells cracking solutionThe myocardial cells were isolated from the hert of the SD rat as early asP1-3with the Multiple joint digestion method.Then we used the method ofpurification based on the different rates of attachment among the various celltypes in order to remove the fibroblas. The myocardial cells were cultured inthe DMEM/F12culture medium containing15%fetal bovine serum.Theworking liquid of Brdu were used to restrain the growth of fibroblasts.Wecollected myocardial cells cracking fluid by repetitive freeze-thawing.3The pVP22-GATA-4/myc-His recombinant plasmid transfection intoBMSCsUsed the transfection reagent, Lipofectamine2000, the pVP22-GATA-4/myc-His recombinant plasmid were transfected into BMSCs.After48h,wedetected the expression of GATA-4with the immune cell chemical dyeingmethod in order to determine the transfection efficiency.Results:124h after noculation, BMSCs attached,which were big and round.After48h, adherent cells were fusiform. Three days later, the proliferation ofBMSCs was significantly, showing a colony sample growth.The cellspresented a variety of forms, mainly for the fibroblasts sample. After7~10days, the cells were spindle and paved with the bottle bottom.24h afterpassage, the cells attached.At first, the cells were round, and gradually to spindle.3days after passage, the cell volume increased obviously. From thesecond generation,the form of BMSCs started to be uniform, showing a fibersample form, which were called "network" or "swirl".Continuous passage.Little change in cell morphology after P2.2The myocardial cells were globate, similar in size and loating in thenutrient solution first. After12h, the cells attached and gradually extendededprocesses.48h later,some individual cells wriggled slowly.48h later, adherentrate reached as high as80%.The great mass of the cells were polygon and heldtogether.Most cells shrinked obviously,with the the frequency of80~120times/min. About7d later, cell clusters formed.Their pulsation was obviouslysynchronism. Immunocytochemistry staining (cTnT) showed the purity ofmyocardial cells reached80%, which met the requirement of experiment.3The A260/280of the plasmid DNA was1.85or so.And theconcentration was600~1000ug/mL. The concentration and purity were all inline with the requirements of the transfection.4On the10th,20th and30th day after induction, we can detect cTnT(cytoplasmic positive expression) expression in the cells of the theexperimental group and the control group,using immunocytochemistrystaining. After statistics analysis,the expression of cTnT gradually increasedWith the passage of time, and there was a significant difference.Theexpression of cTnT in the experimental group was higher than that in thecontrol group On the10th,20th and30th day. statistics analysis showed thatthere was a significant difference between the two groups.Conclusions:1BMSCs differentiated into myocardial cells in protein level in themyocardial microenvironment,which was simulated by the myocardial cellcracking fluid. And the expression increased gradually with the training days.2The differentiation of BMSCs, which were transfected GATA–4,wasmore obviously than the cells whithout transfection in myocardial cellcracking fluid.It may demonstrate that GATA-4gene can promote BMSCs differentiating to myocardial cells. |
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