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Objective To study the expression of L-type calcium channel gene(?1c) and transient outward potassium channel gene(kv4.3)during the procession of MSCs differentiating into cardiomyocytes.Methods MSCs were isolated cultured,passaged,purified,and then exposed to 5-aza for 24 hours.The expression of ?1C and kv4.3 gene was detected by reverse transcription-polymerase chain reaction.Result MSCs had already expressed ?1c and kv4.3 mRNA before 5-aza treatment.The expression of kv4.3 was significantly increased after 5-aza exposure at 24 hoursand at 4th,7th day and 14th day,wheras the expression of ?1C was impaired.Conlusion L-type calcium channel and transient outward potassium channel played important roles during procession of MSCs differentiating into cardiomyocytes.
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Objective To construct the p300 specific siRNA vector in mice and observe its effect on cardiac transcription factor,GATA4,for further study of the effect of p300-mediated histone acetylation on heart growth. Methods Three recombinant plasmid vectors ( p300 RNAi1,p300 RNAi2,and p300 RNAi3s) ,designed and constructed according to the conservative sequence of mice p300,were respectively tansfected into in vitro cultured cardiac muscle cells of suckling mice. Expression of p300 and GATA4 at mRNA and protein levels was detected by RT-PCR and immunofluorescence,respectively. Results The expression level of p300 mRNA was not significantly decreased in pSOS-HUS and p300 RNAi2 groups,but significantly decreased in p300 RNAi1 and p300 RNAi3 groups ( 0. 220 8 ? 0. 020 0 and 0. 170 8 ? 0. 040 0 vs 0. 509 5 ? 0. 030 0,P
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Objective:To explore the method of isolation, purification and culture of rat mesenchymal stem cells (MSCs) in vitro in order to provide an experimental basis for further study.Methods:MSCs were acquired by adhesive screening and density gradient centrifugation methods.The growth of MSCs at different plate densities,different serum concentrations and different culture media were observed and their growth curves were charted.The cell cycle was detected by flow cytometer.Results:The adherent and fibroblast-shaped cells were in good growth state,which were plated at 2?10 9/ml and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and placed in 37℃, 5%CO 2 incubator.Cells grew slowly the first several days after plated,then the growth accelerated at index speed.The results of the cell cycle detection demonstrated that most of MSCs were in G 0G 1 phase.Conclusion:In suitable conditions,MSCs show stable growth and rapid proliferation in vitro.
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Objective:To investigate the ability of mesenchymal stem cells differentiated into cardiomyocytes in vivo.Methods:MSCs isolated form rats bilateral tibias were purified,expanded in vitro culture, labeled with DAPI, then injected into the myocardium of the recipients(including acute myocardial infraction and normal rats).The hearts were harvested 2～4 weeks after implantation.Morphology of the differentiated cardiomyocytes was identified using HE stain and electron microscopy.Cardiac-specific antigen was detected using immunohistochemistry.Results:Viable cells labeled with DAPI can be identified in host myocardium at all time points after implantation.Implanted MSCs showed the growth potential in myocardial environments, after 4 weeks, donor cells derived from MSCs demonstrated myogenic differentiation with the expressing of myosin heavy chain.Donor cells demonstrated cardiomyocytes phenotypes with the expressing of cardiac-specific antigen Cx43.Meanwhile,electron microscopy examination demonstrated the existence of intercalated disks between donor cells and host cells.Conclusion:MSCs can differentiate into cardiomyoytes in host myocardial microenvironments.