GATA-4 Gene Promote Bone Marrow Mesenchymal Stem Cells Induced By 5-azacytidine Differenting Into Cardiomyocytes

Objective:Every year,tens of thousands of patients are diagnosed as"congestive heart failure (CHF)"in the world, which is mostly caused by coronary atherosclerosis and myocardial infarction,Therefore,ischemic heart disease has become a major disease that hazard to human health.Mature myocardial cells are terminally differentiated cells,they can't regenerate once necrosis.Although we can improve symptoms of myocardial ischemia by treating after myocardial infarction (MI) , myocardial necrosis can't be reversed and finally heart failure would happened.The cardiac stem cells in adult myocardium have a small quantity and can not repair the damaged myocardium.As to heart transplantation therapy, there are some restrictions of donor because of the high immunogenicity and expensive cost.So,now many researchers have focused on cell transplantation in the treatment of myocardial necrosis to increase the number of functional cardiomyocytes,further improve the heart function.However, there are still many problems can not be solved in cell transplantation research.The primary of those problems is the low transformation efficiency of cardiomyocytes and could not get numerous contractile cardiomyocytes for cell transplantation.Therefore,it is of great significancestem to research the specific mechanism during the differentiation of myocardial.Bone Marrow mesenchymal stem cells(BMSCs) is easy accessed from the body,and have low immunogenicity;easy to isolated, cultured and expanded in vitro,maintain the undifferentiated state for a long time;have multipotent differentiation potential: under certain conditions they can differentiate into tendon cells,bone cells,cartilage cells,muscle cells,fat cells,fibroblasts,etc.And with high efficiency in the exogenous gene transfection.So,BMSCs are ideal seed cells in tissue engineering . Many people believe that,the processes of the myocardial differentiation begin from the start of the certain or some key genes,and the protein products of these genes are transcription factors,they can make a downstream target genes cascade activated until completion of the differentiation.And GATA-4 is considered particularly close to heart development. It expressed in the early time,involved in differentiation of cardic precursor cell,septation of compartment,cyclization of cordis,normally functional maintenance of conducting system and mature heart.GATA-4 work through its zinc finger and compound with other cardiac-specific transcription factors such as Nkx2-5,TBX5,dHAND and MEF2,or through regulating the expression of genes that control cardiac structure,such asα-myosin heavy chain(α-MHC),cardiac troponin C(cTnC),atrial natriureticp ep tide(ANP)and brain natriureticp ep tide(BNP).Deletion or mutation of GATA-4 can cause abnormal development of the cardiovascular system.GATA-4 can prevent myocardial cell apoptosis by drug-inducing and reduce its toxicity,therefore, it also has good role in normal myocardial cells.cell-penetrating protein(CPP)is a protein that has been shown to be able to penetrate the cell membrane, reach to the nucleus, and can retain its biological activity , in vivo and in vitro experiments,there has successfully delivered bioactive substances such as proteins, peptides , DNA and siRNA to the cells by CPP,VP22 is one of them.Since CPP can be accepted by most types of cells, and can transfer chimeric molecules, people used it to pass the biological activity of high molecular weight protein between the different organizations.The results show the mechanism may be:CPP have strong cationic properties (it is necessary for CPP), led to strong adhesion to cell membrane and nucleotide anions,so it can penetrate the cell membrane and aggregation in the nucleus.So far,CPP is the only successful way in passing chimeric proteins between cells.Liposome transfection method, because of the lack of targeting and low conversion rate,we try to use VP22 to help transfecting GATA-4 to BMSCs.Therefore,we try to use the cell penetrating peptide VP22 and liposome instantaneous transfect GATA-4 gene to SD rat bone marrow mesenchymal stem cells,observed differentiation into cardiomyocytes under the action of 5-azacytidine(5-aza).We try to find the process of cardiac cell differentiation,provide an effective strategy for the stem cells transplantation.Methods:1 The conversion,amplification and identification of eukaryotic expression vector pVP22-GATA-4/myc-HisThe plasmid pVP22-GATA-4/myc-His was presented by Dr. Marc Penn from American.We transformed it into competent E. coli DH5αby the conventional CaCl₂ method,and coated them to the LB medium plate containing Ampicillin,cultured inverted at 37℃for 12²4 h.Picked a single positive bacteria to amplification.Extracted using plasmid extraction kit and detecting concentrations of plasmid,and was identified by XbaI and EcoRI digestion.2 The SD rat bone marrow mesenchymal stem cells isolated, purified and amplifiedBMSCs were isolated from the tibia and femur of SD rats with adherent culture method.BMSCs were at L-DMEM complete mediumcultured with 15% fetal calf serum.The first exchanged of medium at the third day,medium was changed once after 3-4 days.Passaged when cells covered the bottom.We used the third generation of BMSCs for experiment.3 The eukaryotic expression vector pVP22-GATA-4/myc-His transfect BMSCsBefore transfection,our preliminary experiment found the appropriate cell seeding density,the best transfection system.Used the cationic liposome reagent,Lipofectamine 2000,the plasmid pVP22-GATA-4/myc-His were transfected into BMSCs.After 48h,the expression of GATA-4:VP22 fusion protein was detected with immunocytochemical and western blot. 4 5-azacytidine induced BMSCs with exogenous expression GATA-4 gene to differentiate toward cardiomyocyteThe experiment contains three groups, transfected group,untransfected group and blank plasmid group.Transfected group,5-aza induced BMSCs which expressed GATA-4 gene constantly.Untransfected group,5-aza induced BMSCs.Blank plasmid group,which transfected PcDNA 3.1 blank plasmid constantly.10umol/L 5-aza play a role in transfected group,untransfected group for 24h.At 21^th day,28^th day after induction,the expression of GATA-4 and cTnT was detected with immunocytochemical and western blot.5 Statistical analysis:Use SPSS13.0 statistical software.All data showed by mean±standard deviation (x±s).Paired t test is used for statistical analysis. Takeα= 0.05 as testing standards, P <0.05 was considered had a significant difference.Results:1Successful screening, amplificating pVP22-GATA-4/myc-His plasmid.Restriction analysis showed that:get more than 7,500 bp (not digested plasmid) and 1,300 bp (after restriction) of two bands,consistent with the vector component diagrams.2 At primary culture,the cells were colony proliferation.After vaccination,we could see round and refraction of BMSCs , mixed with a large number of blood cells.After 24h,a small amount of BMSCs adhered,which remained round. After 72h, most cells had adhered,they became to polygonal. No adherentig cells cleared by changing fluid gradually. We could see the small colony after 3-4days.After 7-14 days,pieces of adjacent colonies united to form more than 80%,BMSCs had three forms: spindle-shaped fibroblast-like,small round and large flat polygonal.After passaging,the cells began to adhere in 2-4 hours, 24h completely adherented,after 3-5 days those cells merged into pieces.3rd generation later,the cells were uniformed into fibrous, arranged in swirling-like and radial-like.3 The experiment confirmed that the appropriate cell seeding density for transfection was 2×106/cm2,the best transfection system was the plasmid DNA(μg) and Lipofectamine2000 (μl)at a ratio of 1:2.5.After 48h,we detected the expression of GATA-4:VP22 fusion protein through immunocytochemical and western blot in transfected group.There was no positive results in untransfected group and blank plasmid group.4 At 21^th day,28^th day after induction,we detected the expression of GATA-4 and cTnT through immunocytochemical and western blot in transfected group and untransfected group.The expression of GATA-4 and cTnT growed over time,and there had a significant difference(P<0.05).The levels of GATA-4,cTnT expression in transfected group was higher then the untransfected group at 21^th day and 28^th day.Statistical analysis showed that there was a significant difference between transfected group and untransfected group(P<0.01or P<0.05).5 Western blot was detected the expresstion of GATA-4:VP22 in the transfected group at 21 days and 28 days. Untransfected group and blank plasmid group had no positive results.Conclusion:1 With induceing by 5-aza,the myocardial differentiation of BMSCs with exogenous expressing of GATA-4 gene was better than that of non-transfecred BMSCs. It may suggested that GATA-4 gene can promote BMSCs differentiating towards myocardial cells.2 BMSCs with exogenous expression of GATA-4 gene could be differentiated toward cardiomyocytes after 5-aza inducing, and expressed cardiac-specific transcription factors and cardiac-specific genes in the protein level.The expression of the markers was also increased gradually with prolongation of the time.