丹酚酸B联合重组腺病毒修饰对大鼠骨髓间充质干细胞存活和分化的影响  

The Effect of Salvianolic Acid B and Ad-HGF Infection on the Survival and Differentiation of Bone Marrow Mesenchymal Stem Cells

作　　者：邓菊 朱世杰 颜如玉 赵海玉 张建武 DENG Ju;ZHU Shi-jie;YAN Ru-yu;ZHAO Hai-yu;ZHANG Jian-wu(Cardiovascular Medicine Department,Guangzhou Red Cross Hospital Affiliated to Medical College of Jinan University,Guangzhou 510220,China;Department of cardiology,Nanfang Hospital,Southern medical university,Guangzhou,510515)

机构地区：[1]广州市红十字会医院暨南大学第四附属医院心血管内科,广州510220 [2]南方医科大学南方医院心血管内科,广州510515

出　　处：《血栓与止血学》2020年第4期561-565,共5页Chinese Journal of Thrombosis and Hemostasis

基　　金：国家自然科学基金(31700844)。

摘　　要：目的观察丹酚酸B(SalB)联合重组腺病毒Ad-HGF修饰对大鼠骨髓间充质干细胞(MSCs)存活、增殖及向心肌样细胞分化的影响。方法体外贴壁培养法培养MSCs;实验分单纯MSCs组、Ad-HGF组、SalB组和SalB+Ad-HGF组,用AO/EB染色观察细胞的存活,MTT比色法检测细胞的增殖;细胞分单纯MSCs组、5-Aza组、Ad-HGF组、SalB组、5-Aza+Ad-HGF组、5-Aza+SalB组、SalB+Ad-HGF组,分别诱导MSCs,于诱导后第3周观察细胞形态变化,免疫细胞荧光检测心肌细胞特异性蛋白Desmin和Connexin43、Western Blot检测蛋白cTnT的表达。结果培养的MSCs生长良好,AO/EB检测各组细胞生长良好,胞体及胞核正常;MTT提示从第3天开始,Ad-HGF组、SalB组和SalB+Ad-HGF组OD值较单纯MSCs组和5-Aza组显著增高(p<0.05),5-Aza组较单纯MSCs组降低(p<0.05);加入诱导剂诱导3周后,MSCs由梭形逐渐变圆,部分可见肌管样结构;细胞免疫荧光可见心肌细胞特异性蛋白Desmin和Connexin43表达,Western Blot检测见cTnT表达,Ad-HGF转染组和SalB组蛋白表达较单纯MSCs组增强,但较5-Aza组弱,5-Aza+Ad-HGF组、5-Aza+SalB组和SalB+Ad-HGF组较5-Aza组增强。结论SalB和HGF对MSCs的存活无明显损伤影响,可以促进MSCs的增殖和向心肌样细胞分化,两者联合作用效果更明显。Objective To observe the effect of salvianolic acid B(SalB)in combination with recombinant adenovirus Ad-HGF infection on the survival,proliferation,and differentiation into cardiomyocyte-like cells of MSCs.Methods MSCs were separated and purified by attachment culture method;The experiment was divided into 4 groups:MCSs group,Ad-HGF group,SalB group,and Ad-HGF+SalB group.The survival and proliferation of MSCs in different groups were detected by AO/EB staining and MTT assay.To investigate the differentiation of MSCs into cardiomyocyte-like cells,the MSCs were treated by 7 experiment groups for 3 weeks:MSCs group,5-Aza group,Ad-HGF group,SalB group,5-Aza+Ad-HGF group,5-Aza+SalB group,and SalB+Ad-HGF group.After 3 weeks’induction,the changes of the cell morphology were observed by microscope,the expression of cardiomyocyte specific protein desmin and connexin43 were detected by immunofluorescence,cTnT was screened by Western Blot.Results The MSCs were isolated and purified successfully;Cells grew well in each group by the results of AO/EB staining;The MTT assay results showed that the OD value of Ad-HGF group and SalB group was significant higher than 5-Aza group and MSCs group(P<0.05),the OD value of 5-Aza was lower than MSCs group(P<0.05).After induction for 3 weeks,the spindle shape of MSCs became rounder as time went on,and myotube-like structure could be observed in some cells.And the cells differentiated from MSCs were positive for desmin and connexin43 from immunofluorescence results,and positive for cTnT analyzed by Western Blot.The expressions of these proteins were more obvious in Ad-HGF group and SalB group than the MSCs group,but weaker compared with the 5-Aza group,and the 5-Aza+Ad-HGF group,5-Aza+SalB group,and SalB+Ad-HGF group were more distinct than 5-Aza group.Conclusion SalB and Ad-HGF infection could promote the survival,proliferation,and differentiation into cardiomyocyte-like cells of the MSCs in vitro,the combination of SalB and Ad-HGF infection could enhance these biological effects.These

关 键 词：骨髓间充质干细胞 丹酚酸B 肝细胞生长因子 心肌细胞分化 

分 类 号：R542.2[医药卫生—心血管疾病]