小分子水凝胶结合腺病毒介导的肝细胞生长因子基因修饰对大鼠骨髓间充质干细胞生物学特性的影响  

Effects of adenovirus transfection and small molecular hydrogels-mediated hepatocyte growth factor gene modification on biological characteristics of bone marrow mesenchymal stem cells in rats

作　　者：区彩文[1] 陈国钦[2] 张建武[3] 邓菊[3] 吴智业[3] 陈敏生[1] 

机构地区：[1]南方医科大学附属珠江医院心血管内科,广州510280 [2]广州市番禺区人民医院心血管内科 [3]广州医学院第二附属医院心血管内科

出　　处：《中华生物医学工程杂志》2013年第2期89-95,共7页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（31271053、31070856）

摘　　要：目的 观察小分子水凝胶（SMH）结合腺病毒（Ad）介导的肝细胞生长因子（HGF）基因修饰对大鼠骨髓间充质干细胞（MSC）生物学特性的影响.方法 取第9代MSC株进行实验,分为普通培养下MSC对照组（MSC组）、普通培养下转染Ad-HGF的MSC组（Ad-HGF＋MSC组）、在SMH中培养的转染Ad-HGF的MSC组（SMH＋Ad-HGF＋MSC组）、在SMH中培养的MSC组（SMH＋MSC组）.将Ad-HGF转染MSC,48 h后流式细胞仪检测转染率.采用实时定量PCR检测MSC组、Ad-HGF＋MSC组、SMH＋Ad-HGF＋MSC组细胞转染48 h后的mRNA水平.采用ELISA法测定Ad-HGF＋MSC、MSC、Ad-EGFP＋MSC、SMH＋Ad-HGF＋MSC组细胞上清液中HGF蛋白的含量,持续至转染后23 d.普通显微镜观察比较MSC组、SMH＋MSC组、Ad-HGF＋MSC组、SMH＋Ad-HGF＋MSC组细胞在转染继续培养7d后的形态.MTT法检测各组细胞1～7d的生长情况,绘制生长曲线.流式细胞仪检测转染2周后各组细胞表面标志物和细胞周期.各组细胞在5-aza诱导24 h后继续培养3周,免疫荧光化学检测细胞向心肌样细胞分化的能力情况.结果 Ad-HGF转染MSC 48 h后,转染率为74.7％.转染后48 h,MSC组Ct值为14.99;SMH＋Ad-HGF＋MSC组Ct值为8.38;Ad-HGF＋MSC组Ct值为8.51;转染两组均出现明确的HGF基因表达.Ad-HGF＋MSC组、SMH＋Ad-HGF＋MSC两组上清中均有HGF蛋白分泌,Ad-HGF＋MSC组转染后48 h含量达最高峰,为121 μg/L,持续至23 d.SMH＋Ad-HGF＋MSC组HGF含量高峰在第3天,达118μg/L,持续至23 d.MSC组、Ad-HGF＋MSC组细胞均呈成纤维细胞样生长,而转染或未转染Ad-HGF的MSC细胞在SMH水凝胶中三维培养时,细胞多为棒状,一些成球形,细胞之间的分支连接紧密,有聚集生长的趋势.Ad-HGF＋MSC组与MSC组细胞生长曲线相似,各时间点吸光度A值差异无统计学意义,生长趋势吻合.SMH＋MSC组与SMH＋Ad-HGF＋MSC组细胞生长速度前4d与MSC、Ad-HGF＋MSC组比较,吸光度A值差异均无统计学意义;4d后细胞的吸光度AObjective To investigate the effects of adenovirus transfection and small molecular hydrogels （SMHs） mediated HGF on the biological characteristics of bone marrow mesenchymal stem cells （MSCs） in rats. Methods The MSCs of the 9th passage were assigned to he treated with common culture media （group MSC）, Ad-HGF transfection followed by culture in common media （group Ad-HGF＋MSC）, incubation with SMHs alone （group SMH＋ MSC） , and Ad-HGF transfection followed by incubation with SMHs （group SMH＋Ad-HGF＋MSC）. MSCs were transfected with Ad-HGF and the assessment of transfection rate was carried out at 48 h by using flow cytometry. Expressions of HGF mRNA in groups MSC, Ad-HGF＋ MSC and SMH＋Ad-HGF＋MSC were examined by RT-PCR. The supernatant HGF protein of MSCs in groups MSC, Ad- EGFP ＋ MSC, Ad - HGF ＋ MSC and SMH ＋ Ad - HGF ＋ MSC were detected by ELISA till day 23 following transfection. The phase-contrast microscope was employed to compare the MSCs morphology of groups MSC, Ad-HGF＋MSC and SMH＋Ad-HGF＋MSC. MTY approach was adopted to determine the growth of cells in all groups at days 1 to 7 for depiction of growth plots, and flow cytometry was used to assay the cellular surface markers and cell cycles at week 2 following transfection. After 24-h incubation with 5-aza, MSCs of 4 groups were cultured for 3 weeks, and assessment of the capacity of differentiation to cardiac cells was carried out by immune cell fluorescence assay. Results Incubation of MSCs for 48 h yielded a transfeetion rate of 74.7%. At 48 h after transfection , Ct value was 14.99 in group MSC, 8.38 in group SMH＋ Ad-HGF＋MSC, 8.51 in group Ad-HGF＋MSC. There was conclusive HGF gene expression in two transfeetion groups. Apart from group SMH＋Ad-HGF＋MSC, supematant HGF protein was noted in group Ad-HGF＋ MSC, in which the level peaked at 48 h （121 p^g/L） following transfection and sustained to day 23. The supernatant HGF protein peaked at day 3 （118 trg/L） following transfeetion and sustain

关 键 词：小分子水凝胶 腺病毒 肝细胞生长因子 骨髓 间质干细胞 

分 类 号：R3[医药卫生—基础医学]