重组双基因表达质粒pIRES-NGF-VEGF165转染大鼠BMSCs及表达鉴定  

Establishment of recombinant expression of pIRES-NGF-VEGF165 in BMSCs

作　　者：范波胜[1] 娄季宇[2] 白宏英[2] 

机构地区：[1]焦作煤业集团中央医院神经内科,河南焦作454150 [2]郑州大学第二附属医院神经内科,河南郑州450000

出　　处：《中风与神经疾病杂志》2012年第12期1077-1079,共3页Journal of Apoplexy and Nervous Diseases

摘　　要：目的利用脂质体转染技术,将带有神经生长因子(NGF)基因和血管内皮生长因子165(VEGF165)基因的重组双基因表达质粒pIRES-NGF-VEGF165转染大鼠骨髓间充质干细胞(BMSCs),获得能稳定表达NGF和VEGF165的转基因BMSCs。方法采集和培养大鼠BMSCs;利用脂质体转染技术,将pIRES-NGF-VEGF165质粒转染大鼠BMSCs,经G418筛选,获得阳性细胞克隆,进行扩增培养;应用RT-PCR和Western blot检测NGF、VEGF165的表达,并分别利用PC12细胞生长实验和鸡胚绒毛尿囊膜(CAM)血管形成实验进行NGF和VEGF165的活性检测。结果质粒pIRES-NGF-VEGF165成功转染入BMSCs,可正确表达NGF和VEGF165,并具有生物学活性。结论获得了能够稳定表达NGF和VEGF165的大鼠BMSCs,为进一步研究NGF及VEGF165双基因治疗脑梗死奠定了重要基础。Objective To transfect the recombinant plasmid of pIRES-NGF-VEGF165 expressing both nerve growth factor（NGF） and vascular endothelial growth factor 165（VEGF165） into rat BMSCs through liposome transfection technique to obtain the stably recombinant BMSCs which could express NGF and VEGF165 gene.Methods The rat BMSCs were collected and cultured.The constructed plasmid of pIRES-NGF-VEGF165 was transferred into rat BMSCs through liposome transfection technique,and positive clone were obtained and cultured.The expressions of gene NGF and VEGF165 were detected by RT-PCR and Western blot.The activity of NGF and VEGF165 was detected by PC12 cell growth experiment and CAM angiogenesis experiment,respectively.Results The pIRES-NGF-VEGF165 was transfected into BMSCs successfully,through identification by RT-PCR,Western blot and activity assays.Conclusion NGF and VEGF165 were expressed positively in BMSCs,which establishes an important foundation for further study of the NGF and VEGF165 to treat cerebral infarction.

关 键 词：神经生长因子 血管内皮生长因子165 骨髓间充质干细胞 基因表达 

分 类 号：R741.02[医药卫生—神经病学与精神病学]