Researching Of Structure Development During Mesenchymal Stem Cels Differentiation Into Cardiomyocytes In Vitro

Background: With the development of using stem cellstransplantation to treat heart diseases, mesenchymal stem cells (BMMSCs)become the most fascinating scenes because they are easily to be obtained,proliferated, rarely graft versus host disease and rarely ethic or morallimited. Great success has been gained not only cultivation in vitro but alsotransplantation in vivo[1-3]. Our researching group have also uncovered thatBMMSCs can express transcription factor —GATA4, NKx2.5, muscleenhance factor-2C, and transfer growth factor- β after induced by5-azacytidine(5-aza) in vitro[1]. The cardiac functions amelioratedsignificantly after BMMSCs transplanted into rats' dilated hearts[2]. But themechanisms underlying BMMSCs improvement of cardiac function stillneed more analyzing, especially whether BMMSCs can differentiated intocontractile cells in host myocardium or not. As for technical developmentnow, it is hard to observe their contraction directly in beating hearts, so wecan only explain it in side face, that is, blocking the expression of titin invitro—a special structure protein gene of cardiac, then observing whetherBMMSCs can differentiate into cardiac muscle liking cells which containcorresponding structure and contract functions or not. Objective: Constructing recombinant plasmid that can obstruct titinexpression through RNA interference mechanism and transfecting it intoBMMSCs induced by 5-aza. Then observing weather it can block theBMMSCs cardiac-specific differentiation process or not by detectingcardiac structural proteins including titin,myosin heavy chain(MHC),actinand cardiac troponin T (cTnT). Besides, roles of titin in the developing andconstructing of sarcomere is analyzed. Methods and materials:1. Establish a recombinant plasmid vector involving a small interference RNA and transfect it into normal neonatal cardiomyocytes cultured for 1 week. The expression of titin Z band was investigated by immuonofluoresecence.2. Induce BMMSCs with 5-aza; transfect the recombinant plasmid vector mentioned above into them and observe the expression of titin Z band, MHC, actin as well as cTnT by immuonofluoresecence. Results:1. After enzyme-cutting and DNA sequencing, the recombinant plasmid was constructed successfully.2. Expression of titin decreased after recombinant plasmid transfected intoneonatal cardiomyocytes and BMMSCs induced by 5-aza.3. Expression of cTnT was weakened after titin silenced by siRNA. But there were no significant difference in MHC and actin expression. Conclusions 1: BMMSCs can actually be induced into cardiomyocyte-like cells by5-aza in vitro, although the degree of differentiation are still lower and cannot form intact contractive structure basic. 2: And the short hairpin RNA targeting titin N2B region gene exertsrobust inhibition on titin synthesis and forming, the latter affect theexpression of cTNT, but have no influence on myosin and actin. Titin playsa very important role in the process of structure proteins construction.