The Expression Of Cx43/Cx45 After Transplantation Of Bone Marrow Mesenchyma Stem Cells Treated With 5-AZA-2'-Deoxycytidine In The Rats With Acute Myocardial Infarction

Objective:To investigate in vitro isolation,purify and amplification of bone marrow mesenchyma stem cells(MSCs) in rats;whether the MSCs can differentiate to the myocardial-like cells in the presence of 5-Aza-2'-deoxycytidine; to make animal model of acute myocardial infarction(AMI) and observe the expression of Cx43,Cx45 after MSCs treaded with 5-Aza transplantation in rats with AMI.Methods:Bone marrow was sterilely collected from the bilateral thighbone of rat.MSCs were harvested by Percoll method plus adherence method.The MSCs were treated with different concentration of 5-aza and exposed to 5-aza for different time after the stable MSCs line obtained.Sections from cells assemble were analyzed using transmission electron microscope. Immunostaining was performed using antibodies againstα- actin,connexin43, cTnT.180 Wistar rats were randomly divided into four groups:normal control (N),sham operation group(SH),injecting of Sodium Chloride after myocardial infarction(MI),injecting of bone marrow mesenchyma stem cells treated with 5-aza after myocardial infarction(MSCs).The animal model of AMI was established by ligation of the left anterior descending(LAD)coronary artery through thoracotomy.The AMI model was qualified for the elevation of related ST segments of electrocardiogram more than 0.2 mV after operation.The mesenchymal stem cells treated with 5-aza were injected into the infarcted area and border-zone of the infarcted area after 7days in rats of the MSCs group.The rats of MI group were injected with equal Sodium Chloride.The sham group was just under surgery.All the rats were averagely in three branch groups and sacrificed respectively after 4,8 and 12 weeks.To observe the engrafed DAPI-labeled MSCs' migration and distribution in vivi with fluorescence microscope.The heart tissues of nomal area,infarcted area and border-zone of the infarcted area were kept under -80℃.The expression of Cx43,Cx45 in the protein extracts were detected by western blotting(WB).Results:In vitro the MSCs isolated from Wistar rats have favourable reproductive activity and keep the Characteristics of undifferentiation.In the inverted phase contrast microscope we haven't found the myotube-like structure in the MSCs interruptably exposed to 10μmol/L 5-Aza for 48hours,also not found sarcomere and myofilament under the TEM.While we can observe the typical changes in the MSCs persistly exposed to 10μmol/L 5-Aza for 15days and some could show spontaneous beating with the frequency of 15-20 per min. Rich glycogen granules,a number of mitochondria and myofilament were observed.The rate of DAPI-label was 100%in vitro,but the fluorescence intensity would taper with the time lasting.In nomal heart tissues,the expression of Cx43 contents detected with WB among each groups are no difference after intervention for 4,8,12 weeks.The border-zone of the infarcted area in each group:after 4,8 weeks interventions,in MI group & MSCs group,comparing with the normal area,the Cx43 contents decrease in the border-zone of the infarcted area(P＜0.01).Comparing MI group with MSCs group,the expression of Cx43 contents(4,8,12w) in border-zone of the infarcted area in MSCs group is increased(P＜0.05).Comparied with the border-zone of the infarcted area,the Cx43 contents decrease in the infarcted area(P＜0.01).Comparing MI group and MSCs group(8,12w),the expression of Cx43 contents in the infarcted area in MI group is significant reduced(P＜0.05).In nomal heart tissues,the expression of Cx45 contents detected with WB among each groups are no difference after intervention for 4,8,12 weeks.The border-zone of the infarcted area in each group:in MI group(4w),the expression of Cx45 in the border-zone of the infarcted area is more than that of in the normal area(P＜0.01),while no difference in 8,12weeks.Comparing MI group with MSCs group,the expression of Cx45 contents in the border-zone of the infarcted area in MSCs group was less than that of in the MI group(P＜0.05).Comparied with the normal area,the Cx45 contents decreased in the infarcted area(P＜0.01).In MI group(4,8,12w) and in MSCs group(8,12w),the expression of Cx45 in the infarcted area is less than that of in the border-zone of the infarcted area(P＜0.01).There is no difference in the infarcted area between MI group and MSCs group.Conclusions:In vitro MSCs have favourable reproductive activity and keep the Characteristics of undifferentiation.The MSCs could differentiate to the myocardial-like cells in the presence of 5-Aza-2'-deoxycytidine.And the heart function has improved after transplantation of these myocardial-like cells in the model of AMI.AMI reduced the expression of connexin43,connexin45 in the rats' heart,the exogenous cells transplantation could upturn the expression of Cx43 in the core and border-zone of the infarcted area,and down regulate the expression of Cx45 in the border-zone of the infarcted area.No influence on the expression of Cx45 in the core of infarcted area.