摘要
目的:研究Ad.ANG转染骨髓基质细胞(bone marrow stromal cells,BMSCs)的转染效率、产物表达和其表达产物的促血管内皮细胞生长作用。方法:体外培养扩增SD大鼠BMSCs,通过转染β-半乳糖苷酶基因评价腺病毒转染BMSCs的转染效率;通过Western印迹分析和ELISA检测血管生长素(ANG)在细胞内表达和细胞外分泌情况;通过细胞计数的方法研究转染Ad.ANG后的BMSCs培养上清的促内皮细胞生长作用。结果:腺病毒对于BMSCs具有很高的转染效率,转染倍数为50时,转染效率>95％,而且对细胞生长没有影响。Ad.ANG转染BMSCs后可以获得很高的表达水平,第7天达表达高峰[(1 086.808±83.301 pg/ml],15 d后仍可检测到蛋白表达。含有ANG的培养上清与对照组相比具有明显的促血管内皮细胞生长作用(P<O.01)。结论:腺病毒可以安全、有效地转染BMSCs,其携带ANG基因可获得很高的瞬时表达水平,并能促进血管内皮细胞生长。
Objective: To study transfection efficiency, protein expression, and stimulation on vascular endothelial cells proliferation of bone marrow stromal cells (BMSCs) transfected by angiogenin ANG gene mediated by adcnovirus vector ex vivo. Methods: BMSCs were isolated and cultured by gradient centrifugation. The transfection efficiency and cellular toxicity of adenovirus vector to BMSCs were detected by transfection with Ad. LacZ. Expression of Ad. ANG in BMSCs and its secretion in culture medium were measured by Western blot and ELISA. Vascular endothelial cells were incubated with conditional medium containing secreted ANG, and effects on cells proliferation were examined by cell count. Results: Adenovirus transfected BMSCs with a very high efficiency. When MOI was 50, the transfection efficiency was more than 95%. And adenovirus was not harmful for BMSCs growth. When BMSCs were transfected with Ad. ANG, there was a high expression. A maximum expression was observed on the 7th day after transfection [(1 086. 808 ± 83. 301) pg/ml]; ANG were still detectable 15 d later. The conditional medium containing ANG had an excellent ability to stimulate proliferation of vascular endothelial cells than control group (P<0. 01). Conclusion: Adenovirus vector can be transferred into BMSCs efficiently and safely. Adenovirus-mediated ANG gene transfer allows for a high transient expression, and the expressional product can stimulate proliferation of vascular endothelial cells in vitro very well.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2002年第11期1198-1200,共3页
Academic Journal of Second Military Medical University
基金
国家自然科学基金资助项目(30170935).
关键词
血管生长素
腺病毒
骨髓基质细胞
转染
血管内皮细胞
angiogenin
adenovirus
bone marrow stromal cells
transfection
vascular endothelial cells
