| Objectives:Myocardial infarction(MI)is a disease caused by coronary artery occlusion and blood flow interrupted,which local nerosis part of the myocardium occurred because of severe persistent ischemia.More reactive oxygen species(ROS) presented in MI area eventually lead the cardiac myocytes and transplanted cell to apoptosis because of oxidative stress reaction.H2O2 is main ROS.Since the cardiac myocytes are terminally differentiated cells and the injured myocardium can only be replaced by fibrous tissue.Conventional treatment can not reverse the already necrotic cardiomyocytes.The stem cell transplantation in the treatment of myocardial infarction is considered to be promising treatment.Skeletal myoblasts derived from the same mesoderm as cardiomyocytes,which had desirable characteristics self-body transplant characteristics.However,the survival rate of transplanted cells in the host myocardium directly affect the treatment.The experiment confirmed that 70-80% of the transplanted cell would be death three days later after transplantation.The main reason is the apoptosis caused by oxidative stress under the enviroment of ischemia.In recent years a number of studies showed that micro RNAs(mi RNAs) have a regulatory effect on cell proliferation,differentiation,apoptosis and other processes.The role of different mi RNAs are not the same.Studies have shown that mi R-214 has a protective effect on injured myocardium,and has a adjustable role on proliferation and differentiation of normal myoblasts.Therefore,H2O2-injured model of L6 skeletal myobalsts(L6SKM)in vitro was established in this study and exoression of mi R-214 was observed under different injuried conditions.And then the effects and mechanism on proliferation and apoptosis of mi R-214 transfected L6 SKM under oxidative stress were studied.Methods:1 Effect of mi R-214 on the proliferation of L6SKML6SKM were cultured and were divided into five groups(normal control group,pre-mi R-214 group,pre-scramble group,anti-mi R-214 group,anti-scram- ble group.The expressions of mi R-214 in each group were detected by real-time quantitative PCR(q PCR).The vitality of L6 SKM were detected with MTT assay.The expressions of PCNA and cyclin D1 were detected by immunocytochemistry and the cell cycle was analysed by flow cytometry.2 The expression of mi R-214,myo D,myogenin in L6 SKM during differential cultur at different timeL6SKM were cultured in vitro and then in 2% horse serum for 0 day,2 d- ays,4 days,6 days when the cell fused to 60-70%.The expressions of mi R-214, myo D,myogenin was determined by real-time quantitiative PCR(q PCR).3 The expressions of mi R-214,myo D,myogenin in L6 SKM during differential culture at different time after mi R-214 transfectionL6SKM were divided into five groups(normal control group,pre-mi R-214 group,pre-scramble group,anti-mi R-214 group,anti-scramble group).The L6 SKM were cultured in medium with 2%horse serum for 0day,2days,4days after mi R-214 transfection.The expression of mi R-214,myo D,myogenin were determined by real-time quantitative PCR(q PCR).4 Effect of H2O2 with different concentration and at different time on L6SKML6SKM were divede into normal control group,H2O2 action group(H2O2 concentration is 50μmol/L,100μmol/L,200μmol/L,300μmol/L,400μmol/L,600 μmol/L;reaction time was 4h).The cell viability of L6 SKM in each group were measured by MTT and the expression of mi R-214 was measured by q PCR. L6 SKM were cultured in medium with 300μmol/L H2O2 at different times(0.5 h,1h,3h,4h,5h,7h).The viability was analysed by MTT assay and the express- ion of mi R-214 was measured by q PCR.5 Effect of mi R-214 on proliferation and apoptosis of H2O2-injured L6 SKM.L6SKM were transfected with mi R-214 in good condition and then cultured in medium with 300μmol/L H2O2 for 4h.The cells were divided into six groups respectively(normal control group,H2O2 group,pre-mi R-214+H2O2 group, pre-scramble+H2O2 group, anti-mi R-214+H2O2 group, anti-scramble+ H2O2 group).The activity of L6 SKM were detected by MTT and the expressio- ns of PCNA and cyclin D1 were detected by immunohistochemistry.The cell cycle and apoptosis rate were measured by flow cytometry.The expression of caspase-3,Bcl-2,BAX were detected by western blotting.Results:1 Effect of mi R-214 on the proliferation of L6SKMq PCR results showed that the expression level of mi R-214 in pre-mi R-21 4 group was significantly higher than the normal control group.Anti-mi R-214 could inhibit the expression of mi R-214.There was no different among the normal control group,pre-scramble group and anti-scramble group.The results indicated the transfection was successful.MTT cell viability assay showed that the cell viability in pre-mi R-214 group was significantly increased than normal control group and was lower in anti-mi R-214 group than normal control group.There was no different among the normal control group,pre-scramble group and anti-scramble group.Flow cytometry analysis results showed that PI values in pre-mi R-214 group was higher than normal control group,anti-mi R-214 group was lower than normal control group.Immunocytochemistry assay showed that the expression of PCNA and cyclin D1 in pre-mi R-214 group was higher than normal control group,anti-mi R-214 group was lower than normal control group. The result showed that mi R-214 can promote L6 SKM proliferation in growth medium.2 The expressions of mi R-214,myo D,myogenin in L6 SKM during differentiatial cultured at different timeq PCR results showed that the expressions of mi R-214,myo D,myogenin were gradually increased with extenden differentiatial cultured time.3 Effect of mi R-214 on the expression of mi R-214,myo D,myogenin in L6 SKM after differentiatial culture at different timeq PCR showed that the expression of mi R-214,myo D,myogenin in pre-mi R-214 group were higher than normal control group,and were increased with extended time.The expression of mi R-214,myo D,myogenin in anti-mi R- 214 group were lower than normal control group.It showed that mi R-214 can promote L6 SKM differentiation in differentiatial medium.4 Effect of H2O2 on L6SKMMTT results showed that the cell viability of L6 SKM decreased with the increase concentration of H2O2 and the extension of time.This effect was almost dose and time dependent.We finally selected the 300μmol/L concentration of H2O2 which cell survival rate is about 50%,established model of the oxidative stress.q PCR test result showed that the expression of mi R-214 in H2O2 group were gradually increased compared with the normal control group.And with the increase of H2O2 concentration and time,the expression of mi R-214 were gradually increased.5 Effect of mi R-214 on proliferation and apoptosis of H2O2-injured L6SKMMTT result showed that the cell activity in pre-mi R-214+H2O2 group was significantly higher than H2O2 group and was lower in anti-mi R-214+H2O2 group than that in H2O2 group.There was no difference among the normal control group,pre-scramble group and anti-scramble group.Immunocytochem- Istry assay showed that the expression of PCNA and cyclin D1 in pre-mi R-214+H2O2 group was higher than H2O2 group,anti-mi R-214+H2O2 group was lower than that in H2O2 group.Flow cytometry results showed that the PI values in pre-mi R-214+H2O2 group was higher than H2O2 group,while anti-mi R-214+H2O2 group was lower than that in H2O2 group.The apoptosis rate in pre-mi R-214+H2O2 group was lower than H2O2 group,while anti-mi R-214+H2O2 group was higher than that in H2O2 group.Western blotting results showed that the expressions of capase-3 and BAX in pre-mi R- 214+H2O2 group were lower than H2O2 group,the expression of Bcl-2 in pre- mi R-214+H2O2 group was higher than H2O2 group.The anti-mi R-214+H2O2 group was contrary.There was no difference among the normal control group,pre-scramble group and anti-scramble group.Conclusion:1 mi R-214 can promote L6 SKM proliferation in growth medium,and promote differentiation in differentiation medium.2 H2O2 could coursed increased expression of mi R-214 in L6 SKM and this effect was almost dose and time dependent.3 mi R-214 could promote proliferation and inhibit its apoptosis of H2O2 –injured L6 SKM.mi R-214 could inhibit its apoptosis by reducing the expression of capase3 and BAX and increasing the expression of Bcl-2. |
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