bFGF基因体外转染缺血性心肌病大鼠骨髓基质干细胞研究

Transfection of bFGF Gene into Bone Marrow Stromal Cells of Ischemic Cardiomyopathy Rats in Vitro

目的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)是存在于骨髓等多种组织中的具有形成骨、软骨、脂肪、神经及成肌细胞的多种分化潜能的一类细胞群。该研究将碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)转染入BMSCs对缺血性心肌病(ischemic cardiomyopathy,ICM)的修复作用及机制,为应用bFGF修饰的BMSCs进行移植治疗ICM的动物实验研究提供理论基础。方法采用左前降支结扎法对大鼠进行ICM模型的制备。造模成功后,提取及体外培养大鼠BMSCs,并绘制其生长曲线。首先通过病毒转染的方法将bFGF基因转染入大鼠BMSCs,采用免疫组化法、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)法以及免疫荧光法检测b-FGF基因转染大鼠BMSCs的效果及表达结果,CCK-8法检测大鼠BMSCs增殖活性变化。结果ELISA检测不同时间点转染上清液中分泌的bFGF含量,结果在12~60 h内浓度呈现上升趋势,在60 h可达(19.702±0.074)ng/mL。与对照组比较,转染组分泌的bFGF在72 h内含量增加且均差异有统计学意义(t=122.808,P<0.05)。转染后的部分细胞形态发生变化。免疫荧光法和免疫组化均证实在胞浆中有bFGF颗粒着色阳性,转染成功率可达(72.19±8.83)%,转染效率较高。CCK-8法测大鼠BMSCs细胞增殖活力,结果显示与对照组相比,转染组增殖活性均明显增加,增殖率可高达(124.3±1.2)%(t=23.951,P<0.05)。结论通过病毒转染的方法,成功将具有多重生物学效应的bFGF基因转染入大鼠BMSCs。转染后的大鼠BMSCs可在较长时间内保持较高浓度的bFGF的分泌。bFGF基因转染可促进大鼠BMSCs的增殖和分化。

Objective Bone marrow mesenchymal stem cells(BMSCs)are a group of cells that exist in bone marrow and other tissues and have the potential to differentiate into bone,cartilage,fat,nerve and muscle cells.In this study,basic fibroblast growth factor(bFGF)was transfected into BMSCs for the repair of ischemic cardiomyopathy(ICM)and its mechanism,providing a theoretical basis for animal experimental studies on the transplantation of bFGF modified BMSCs for ICM.Methods Rats were prepared with ICM model by left anterior descending ligation.After successful modeling,rat BMSCs were extracted and cultured in vitro,and their growth curves were drawn.First,the bFGF gene was transfected into rat BMSCs by virus transfection,and the b-FGF gene was transfected by immunohistochemistry,enzyme-linked immunosorbent assay(ELISA)and immunofluorescence.The effect and expression of mouse BMSCs were detected by CCK-8 method.Results ELISA was used to detect the content of bFGF secreted in the supernatant of transfection at different time points.The concentration showed an increasing trend within 12-60 h,and reached(19.702±0.074)ng/mL at 60 h.Compared with the control group,the content of bFGF secreted by the transfection group increased within 72 h and there was statistically significant difference(t=122.808,P<0.05).The morphology of some cells after transfection changed.Both immunofluorescence and immunohistochemistry confirmed that bFGF particles were positively stained in the cytoplasm,and the transfection success rate was (72.19±8.83)%, and the transfection efficiency was higher. The proliferationof rat BMSCs was detected by CCK-8 method. The results showed that compared with the control group, the proliferationactivity of the transfected group was significantly increased, and the proliferation rate was as high as (124.3±1.2)% (t=23.951,P<0.05). Conclusion The bFGF gene with multiple biological effects was successfully transfected intorat BMSCs by viral transfection. Transfected rat BMSCs maintained a higher concentration of bFGF secretion for alonger period of time. bFGF gene transfection can promote the proliferation and differentiation of rat BMSCs.