Research On The Role Of Biomimetic Electrical Stimulation In The Differentiation Of Rat Bone Marrow Mesenchymal Stem Cell Into Cardiac Phenotype Inducedby Microenvironmental Factors Of Cardiomyocyte In Vitro

[Objective]Discuss the role of biomimetic electrical stimulation in the differentiation of rat bone marrow mesenchymal stem cell into cardiac phenotype.[Methods]1. The isolation cell were primary cultured and divided into difference groups according to implant denstity , first medium change , difierent concentration of fetal bovine serum,medium PH , after ten days collect the cells and count it , their growth curves werecharted by MTT ways, immuno-histochemistry were used to evaluate the expression of the cell CD44,CD34 , and the cell cycle was detected by flow cytomete.2. Neonatal rats'cardiac myocytes were isolated to be cultivate by two different combination of trypsin and collagenase II (way-1 and way-2 ) , comparing the quantity and livability , moreover, observing the change of Morphocytology and detecting the activity ,immuno fluorescence were used to evaluate the purity of the cell.3. MSCs and cardiac myocytes from SD rats were isolated and,moreover, MSCs were co-culture with cardiac myocytes (MSCs/ cardiomyocyte co-culture system). The co-culture system were divided to stimulated group and non-stimulated group,and every groups divided by incubated for 3,5 and 7 days were named as subgroupⅠ,ⅡandⅢrespectively.MSCs of stimulated group were stimulated for 2 h every day by supra-threshold square biphasic pulses with 5ms duration,0.5 Hz,10V(current intensity of 20μA). inducing.Light microscope were used to identify the influences of characteristic morphological of MSCs in every subgroup,immuno -fluorescence was used to detected the expression of cTnT in MSCs.4. MSCs of the second passage was used ,it was divided into stimulated group(intervention group) and non-stimulated group(control group) . And the stimulated groups was divided by voltage intensity of 5,10,20 (current intensity of 10,20,40μA respectively), which were named as subgroupⅠ,ⅡandⅢrespectively. MSCs of stimulated group were stimulated for 2 h every day, persistence for 7days . After 7 days,And then, immuno -fluorescence was used to detected the expression of cTnT or not in every group.[Results] 1. The adherent fibroblast-shaped in good growth state, the reasonable cell inplant denstity is 5×105/cm2, first medium change time is 48h, difierent concentration of fetal bovine serum is 10%,medium PH is 7.2. The growth curves show that the cell growth slowly the first several days ,then growth accelerated at index speed,after that growth stable state. The membrane of MSCs expression of the cell CD44, not CD34. The results of the cell cycle detection demonstrated that most of MSCs were in G0/G1 phase.2. Cardiomyocytes with a good quantity and livability were obtained by two different combination ways, and the livability of way-2 is higher than way-1. The cardiac myocytes can adhere and impulse well . immunofluorescence stain confirmed the cell with purity higher than 95%.3. The growth of stimulated group was better than that of nonstimulated group . In stimulated subgroupⅡ, cTnT were detected expression in some cells , but not that be observed in no-stimulated groupⅡ.There were detected the expression of cTnT in MSCs both of the stimulated subgroupⅢand no-stimulated groupⅢ,however, the expression of cTnT in stimulated subgroupⅢwere higher than that of non-stimulated subgroups respectively.4. The MSCs was Electrical stimulated for 7days, there was not expression of cTnT in Electrical stimulated group with voltage intensity of 5,10,20 ,and control group after immunofluorescence stain.[Conclusion] 1. Biomimetic electrical stimulation could redound differentiation of rat bone marrow mesenchymal stem cells(MSCs)to cardiomyocyte induced by microenvironmental factors of cardiomyocyte in vitro, the time exhibition of cardiomyocyte-like cell were earlier , and the efficiency of different- -tiation rate were higher.2. In the parameter of our empirical study, biomimetic electrical stimulation solitary can not be enough to differentiate rat bone marrow mesenchymal stem cell into cardiac phenotype .