Effects Of Gene Nkx2.5, Rehmannia Glutinosa Oligosaeeharides And K_ATP Opener On The Trans-differentiation Of Bone Marrow Mesenchymal Stem Cells Into Cardiomyocytes

Background: Heart failure is a clinical syndrome caused by ventricular dysfunction. Itcauses reduced cardiac output, increased ventricular pressure, causing breathingdifficulties, fatigue, edema and other symptoms. At present the treatment of heartfailure includes drug treatment, auxiliary device, heart transplantation, celltransplantation and gene therapy. Although drug and device play an important role inthe treatment of heart failure, but patients with heart failure have not got the basicimprovement. Stem cells transplantation of great concern can add functional cells torepair the functional cardiomyocytes loss in heart failure. The unique properties ofMSCs （easily isolated and amplified from the BM, their multi-lineagepotential,immunologically tolerated as an allogeneic transplant）and have led to theirintense investigation as a cell-based therapeutic strategy for cardiac repair. Althoughthe efficacy of MSCs in treating heart failure has been confirmed，the survival rate andconversion rate of the stem cells after transplantation are extremely poor. How toinprove the MSCs survival and differentation is the major problem of cell theropy..Objective: To study the effect of gene Nkx2.5, Rehmannia glutinosa oligosaccharide（RGOs） and ATP sensitive potassium channel (K_ATP) opener on the proliferation of ratBM-MSCs and its differentiation into cardiomyocytes.Methods: BM-MSCs of4weeks old male SD rats were isolated and amplified in vitro,then infected by lentivirus carrying Nkx2.5gene. BM-MSCs and Nkx2.5+BM-MSCswere preconditioned by RGOs and K_ATPopener. All the cells were divided into6groups: BM-MSCs, Nkx2.5+BM-MSCs, RGOs+BM-MSCs,Nkx2.5+RGOs+BM-MSCs,pinacidil+BM-MSCs and diazoxide+BM-MSCs. The anterior four groups were tested the proliferation and all the groups were induced apoptosis by H₂O₂and then detect theapoptosis rate by flow cytometry. The total mRNA of BM-MSCs transfected for2,3,4,6weeks, BM-MSCs predictioned by K_ATPopener for2hous and Nkx2.5+BM-MSCspredictioned by RGOs for3weeks were extracted to detect the expression cardiacspecific gene TnI, CK, CKMB, Cx43and α-MHC. Protein of BM-MSCs transfectedfor3weeks and predictioned by K_ATPfor2h were extracted to detect the expression ofCK, CKMB and TnI.Results: There was no significant difference in the proliferation of the anterior fourgroups. Nkx2.5could decreased the apoptosis rate from66.9%to51.7±8.4%. Therate in RGOs group decreased from66.9±7.3%to38.9±3.2%, RGOs+Nkx2.5group36.1±2.8%, pinacidil37.7±3.8%and diazoxide21.6±1.8%. BM-MSCs transfected byNkx2.5could express the cardiac-special gene CK, α-MHC and Cx43.RGOs+Nkx2.5+BM-MSCs group also expressed CK and α-MHC,but there was nodifferences between with Nkx2.5+BM-MSCs group. BM-MSCs predictioned bypinacidil and diazoxide express TnI, CK and Cx43. TnI increased16.8±3.7and11.5±2.6times relative to the control, CK15.0±4.0and4.3±0.4times, Cx432.5±0.46and2.9±1.21times. BM-MSCs transfected for3weeks and predictioned by diazoxidefor2h expressed protien of CK and CKMB.Conclusion: Nkx2.5could increased the expression of cardiac special gene inBM-MSCs. RGOs could not improve the differentation, but could increase thesurvival rate of BM-MSCs induced apoptosis by H₂O₂. K_ATPopener could inducedBM-MSCs differentiate to cardiomyocytes and improve the resistance to apoptosis.