人血管内皮生长因子165基因转染对C17.2神经干细胞体外增殖分化的影响  

Effects of vascular endothelial growth factor 165 gene transfer on proliferation and differentiation of C17.2 neural stem cells in vitro

作　　者：沈庆煜[1] 王艺东[1] 李梅[1] 黎祥喷[1] 吕瑞妍[1] 肖颂华[1] 王莉梅[1] 邢诒刚[1] 

机构地区：[1]中山大学附属第二医院神经内科,广东省广州市510120

出　　处：《中国临床康复》2005年第13期50-53,共4页Chinese Journal of Clinical Rehabilitation

基　　金：广东省卫生厅科研基金资助项目(B2004047)~~

摘　　要：目的:构建含有人血管内皮生长因子165基因的重组腺病毒载体,并观察其对C17.2神经干细胞体外增殖和分化的影响。方法:将血管内皮生长因子165基因克隆至腺病毒穿梭质粒pAdTrack-CMV,经酶切线性化后,与腺病毒骨架质粒pAdEasy共同转染大肠杆菌BJ5183同源重组获得重组腺病毒质粒,将线性化的重组腺病毒质粒转染293细胞包装获得有感染能力但复制缺陷的重组腺病毒pAdCMV血管内皮生长因子165,转染体外培养的C17.2神经干细胞。以单纯携带绿色荧光蛋白腺病毒转染为对照1组。通过酶联免疫吸附测定检测其在C17.2神经干细胞中不同时间段的表达。C17.2神经干细胞培养24h后,将pAdCMVGFP和pAdCMV血管内皮生长因子165转染C17.2神经干细胞(pAdCMV绿色荧光蛋白和pAdCMV血管内皮生长因子165转染组),24,48,72h后每孔加入四氮唑盐10μL,检测pAdCMV转染对C17.2神经干细胞增殖的影响。将未进行pAdCMV绿色荧光蛋白,pAdCMV血管内皮生长因子165转染C17.2神经干细胞设为对照2组。收集C17.2神经干细胞(C17.2神经干细胞组)和已经转染pAdCMVVEGF165的C17.2神经干细胞(pAdCMV血管内皮生长因子165的C17.2神经干细胞组),对两组细胞进行培养,将上述细胞爬片进行多聚甲醛固定,行巢蛋白和神经元特异性烯醇化酶免疫组化法检测。结果:①重组腺病?AIM:To establish the recombinant adenovirus vector containing vascular endothelial growth factor165 (VEGF165), and observe the effect on proliferation and differentiation of C17.2 neural stem cells in vitro. METHODS: VEGF165 gene was cloned to pAdTrackCMV, and then the constructed plasmid was treated with endonuclease, and transferred to BJ5183 Escherichia coli combined with pAdEasy to obtain the adenovirus plasmid. Following treatment with endonuclease again, the developped linear recombinant adenovirus plasmid was transferred and packaged in 293 cells, and recombinant adenovirus containing VEGF165 gene (AdCMV VEGF165) was finally obtained. After the infection of AdCMV VEGF165 to C17.2 neural stem cell in vitro, adenoviruses only contained Green Fluorescent Protein(GFP) were selected as control group 1.Enzyme linked immunoadsordent assay(ELISA) was used to evaluate the expression of VEGF165 in infected cells at different time points. After 24 hour culture of C17.2 cells, pAdCMV GF and pAdCMV VEGF165 were transferred to C17.2 cells (pAdCMV GFP and pAdCMV VEGF165 group), 10μL MTT was added into each pore after 24, 48 and 72 hours,and then the effect of pAdCMV transfection on proliferation of C17.2 neural stem cells was detected. The non transferred C17.2 neural stem cells with pAdCMV GFP and pAdCMV VEGF165 were selected as control group 2. C17.2 neural stem cells (C17.2 neural stem cells group) and transferred C17.2 neural stem cells with pAdCMV VEGF165(pAdCMV VEGF165 group) were collected for culture.The above mentioned cellular slides were fixed with citromint and were detected with immunohistochemistry staining of nidogen and neuron specific enolase (NSE). RESULTS:①Recombinant AdCMV VEGF165 was packaged successfully in 293 cells, the titer of virus was 2×1011 efu/L. And VEGF165 was expressed stably in infected C17.2 neural stem cells, which was(710±82)ng/L on the third day, and significantly higher than before infection[(118±20)ng/L], reached to the peak on the fifth [(1 110±126) ng/L] and seventh

关 键 词：干细胞 内皮生长因子/遗传学 转染 神经元 

分 类 号：R743[医药卫生—神经病学与精神病学]