| 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. |
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