Salvia Combined With Bone Marrow Mesenchymal Stem Cell Transplantation For Collagen Remodeling And Cardiac Function In Rabbits With Acute Myocardial Infarction

Popurse:To establish a method of in vitro isolation,purification and amplification of bone marrow derived mesenchymal stem cells of adult rabbits for a further study.Methods:Bone marrow tissue was harvested from healthy Japanese rabbits.Mesenchymal stem cells were isolated by the method of density gradient centrifuge.Then MSCs were proliferated by adhering culture.CD34,CD29 antigen of MSCs were identified by flow cytometry.At the third passage, MSCs were labeled with BrdU(5-Bromo-2'-deoxy-uridine) and the rate of lable was measured by immunohistochemical method.Results:Primary cultured MSCs start adhering to plates 48 hours after seeding,and spindle-shaped MSCs were observed under microscopy.8-10 days after seeding MSCs were homogenous population and exhibited a spindle-shaped or triangle-shaped.Confluence of MSCs reach to 80%and growing at a rapid speed,MSCs were exhibited a whirlpool-shaped.CD34,CD29 were directed by flow cytometry.Cultured MSCs after incubating of BrdU,the rate of in vitro labling was 80%.Conclusion:In this study,MSCs were isolated and proliferated stably and rapidly.Cultured MSCs possessed high activity of growing and amplification, and were appropriate for further research of transplantation in myocardial infarction. Popurse:To observe the effects of miltiorrhiza combined with marrow mesenchymal cells(MSCs) transplantation on the collagen matrix remodeling after acute myocardial infarction in rabbits,and investigate the synergetically therapeutic effects of miltiorrhiza and MSCs transplantation.Methods:MSCs were harvested from bilateral femur of two rabbits using the method of density gradient centrifuge isolation and adhering culture.At the third passage,MSCs were labeled with Brdu(5-Bromo-2'-deoxy-uridine).Ten rabbits served as normal control group,and other 60 rabbits were used for establishing models of acute myocardial infarction.After model establishment, rabbitts were randomly divided into 4 groups,model group,MSCs group, miltiorrhiza group,and MSCs and miltiorrhiza combition group.2×10~6MSCs labeled Brdu were injected into the vein of the MSCs group and MSCs and miltiorrhiza after seven days of successful models.Miltiorrhiza 15.4mg/kg were injected into the vein of the miltiorrhiza group and combition group after successful models,once a DAY for ten days.Light and electron microscope were taken to observe twenty-eight days after cellular transplant respectivly.The collegen volum fraction(CVF) and the ratio of typeⅠandⅢcollegen were measured by means of masson and immunohistochemistry.The levels of serum matrix metalloproteinase enzyme-1(MMP-1),matrix metalloproteinase enzyme inhibitor -1(TIMP-1),and levels of typeⅠandⅢcollegen were detected by means of enzyme linked immunoadsorbnent. Results:MSCs by vein-injection labled Brdu were found in the myocardial infarction and marginal zones in MSC group and combition group after twenty-eight days of AMI.In myocardial infarction groups,by light microscope,a large number of myocadial fibration were found in the infarcted zone.In stem cell groups, a great number of inflammatory cells were found in the infarcted and marginal zone.In stem cell groups,by electron microscope,a great number of fibroblast were found in the infarction zone.Compared with the model group,the levels of serum typeⅠcollegen was significantly increased(P<0.01) in the MSCs group and MSCs and miltiorrhiza combition group,the levels of serum typeⅢcollegen was increased but there was no difference in each group(P>0.05).Compared with the model group,the levels of serum MMP-1 decreased and TIMP-1 increased in MSCs group,miltiorrhiza group,and MSC and miltiorrhiza combition group,but there was no difference in each group(P>0.05).Compared with the normal group, CVF significantly increased(P<0.05) and the ratio of typeⅠandⅢcollegen decreased significantly(P<0.05)in model group,Compared with the model group, CVF decreased significantly(P<0.05) in MSC group,miltiorrhiza group,but combition group was better(P<0.05),the ratio of typeⅠandⅢwas significantly increased(P<0.05),and combition group was more significant than MSCS group.Conclusion:1.The model of myocardial infarction in rabbits was build successfully by ligaturing left anterior descending coronary artery 2.MSCs by vein transplantation were found in the myocardial infarction and marginal zones.3.MSCs transplantation can improve the ratio of typeⅠandⅢcollegen and inhibit the collagen matrix remodeling after acute myocardial infarction in rabbits.4.Miltiorrhiza can inhibit the collagen matrix remodeling of AMI rabbits,and has a better effect combined with MSC transplantation,and with synergetically therapeutic effects on collagen remodeling. Popurse:To observe the effects and mechanism of miltiorrhiza combined with marrow mesenchymal cells(MSCs) transplantation on cardiac function after acute myocardial infarction in rabbits,and investigate the synergetically therapeutic effects of miltiorrhiza and MSCs transplantation.Methods:Mscs were harvested from bilateral shin of two rabbits using the method of density gradient centrifuge isolation and adhering culture.At the third passage,MSCs were labeled with Brdu(5-Bromo-2'-deoxy-uridine).Ten rabbits served as normal control group,and other sixty rabbits were used for establishing models of acute myocardial infarction.After model establishment, rabbitts were randomly divided into four groups,model group,MSCs group, miltiorrhiza group,and MSC and miltiorrhiza combition group.2×10~6MSCs labeled Brdu were injected into the vein of the miltiorrhiza group and combition group after successful models.Miltiorrhiza 15.4mg/kg were injected into the vein of the miltiorrhiza group and combition group after successful models,once a day for ten days.Doppler echocardiography was used to detect left ventricular internal dimension in diastolic(LVDD),fractional shortening(FS) after twenty-eight days of successful models;Myocardium apoptosis was observed at infracted and marginal regions by TUNEL staining.NF-kb,bcl-2,fas,fasL were measured by fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and western blot methods after thirty-five days of successful models.Results:Compared with the model group,left ventricular end-diastolic diameter were significantly decreased(P<0.01) and shortening fraction were significantly increased(P＜0.01)in the MSCs group,miltiorrhiza group,and MSC and miltiorrhiza combition group.The changes were better in the miltiorrhiza combition group than the MSC group(P<0.05).Compared with the model group, myocardium apoptosis were significantly decreased(P<0.01) in the MSC group, miltiorrhiza group,and MSCs and miltiorrhiza combition group,and combition group was better than MSC group.It was founded that NF-kb,fas,fasL protein expression increased and bcl-2 protein expression decreased in the model group; NF-kb,fas,fasL protein expression were significantly decreased(P<0.01) in the MSC group and combition group,and combition group were more significant than MSCs group.Bcl-2 expression was increased in the miltiorrhiza group,but other groups were not.Conclusion:1.MSCs transplantation by vein injection can improve cardiac function of AMI rabbits.2.MSCs transplantation can reduce the expression of fas,fasL and NF-kb,which were related to apoptosis and inflammation.3.The improvement of cardial function by MSCs transplantation could be related to the inhibition of apoptosis and inflammation.4.Miltiorrhiza can improve cardiac function of AMI rabbits,and has a better effect combined with MSC transplantation,and with synergetically therapeutic effects on myocardial infarction.