脑源性神经营养因子转基因成纤维细胞的构建与检测  

Construction and detection of the fibroblasts modified with brain-derived neurotrophic factor gene

作　　者：孟步亮[1] 李力燕[2] 徐丹[2] 刘佳[2] 王廷华[2] 

机构地区：[1]昆明医学院人体解剖学教研室,昆明650031 [2]昆明医学院神经科学研究所,昆明650031

出　　处：《神经解剖学杂志》2011年第1期110-114,共5页Chinese Journal of Neuroanatomy

摘　　要：目的:构建大鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因修饰的成纤维细胞(fibroblasts FBs)。方法:真核表达载体pcDNA3/BDNF由昆明医学院神经科学研究所提供。FBs取自胎鼠。pcDNA3/BDNF重组质粒载体以阳离子脂质体LipofectamineTM2000介导转染FB,并统计转染率。应用BDNF抗体进行免疫细胞化学和Western Blot检测以鉴定BDNF在细胞内的表达。并在体外培养条件下,应用经转染FBs和未转染FBs的培养液上清培养肾上腺嗜铬细胞瘤(pc12)细胞,验证BDNF的生物活性。结果:胎鼠成纤维细胞体外培养生长状况良好。FBs转染率为(52.14±3.31)%。免疫细胞化学和Western Blot结果表明BDNF在FBs中过表达,转染组与未转染组相比BDNF阳性细胞更多,染色更深,BDNF表达量明显增多,且具有促进PC12细胞生长的活性。结论:上述结果表明,大鼠源性BDNF基因修饰的FBs构建成功。Objective：To construct the fibroblasts（FBs） mordified with brain-derived neurotrophic factor（BDNF）gene.Methods：The recombinant vector（pcDNA3/BDNF）was supplied by Institute of Neuroscience of Kunming Medical College.FBs were taken from the embryo of rats.FBs were transfected with pcDNA3/BDNF using LipofectamineTM 2000 transfection reagent.The transfection efficiency has also been statisticed.The expression of BDNF was analyzed by immunocytochemistery and Western Blot.PC12 cells cultured with the supernatant of transfected fibroblasts and control culture fluid.The effects of culture medium of BDNF-gene modified fibroblast on the PC12 cells in vitro were observed.Results：The FBs taken from the rats＇embryo grew well.The transfection efficiency of FBs was（52.14±3.31）%.The immunocytochemistery and Western Blot showed that the BDNF was expressed successfully in fibroblasts.There were more BDNF positive cells in transfected FBs than that in non-transfected FBs,and the expression of BDNF in transfected FBs was also higher than that of the control group.The culture medium of BDNF-gene modified fibroblast could promote the PC12 cells growth in vitro.Conclusion：These results showed that FBs modified with BDNF gene were constructed successfully.

关 键 词：脑源性神经生长因子 成纤维细胞 转染 

分 类 号：R651.2[医药卫生—外科学]