CAJ | 학술논문

目的探讨重组腺病毒介导血管内皮生长因子(VEGF)165基因治疗对缺氧状态下对神经干细胞凋亡的作用.方法体外培养C17.2神经干细胞,建立神经干细胞缺氧模型.将携带人类VEGF基因的重组腺病毒感染C17.2神经干细胞,Western-blotting分析VEGF蛋白的表达,并用流式细胞术测定细胞凋亡率的变化.Hoechst33342染色荧光显微镜观察凋亡小体.结果转染pAdCMVVEGF165的C17.2神经干细胞成功表达VEGF蛋白;缺氧后神经干细胞凋亡率为(19.98±0.55)%,而转染pAdCMV VEGF165后的细胞凋亡率为(10.38±0.48)%(P<0.01),转染pAdCMV VEGF165+VEGF反义寡核脱氧核酸经干细胞凋亡率为(19.07±0.64)%,与对照组无显著差异(P>0.05).结论腺病毒介导的VEGF165基因能高效表达VEGF;外源性VEGF对C17.2神经干细胞具有抗凋亡作用,能够提高C17.2神经干细胞对缺氧的耐受性,有利于神经干细胞的生存.
목적탐토중조선병독개도혈관내피생장인자(VEGF)165기인치료대결양상태하대신경간세포조망적작용.방법체외배양C17.2신경간세포,건립신경간세포결양모형.장휴대인류VEGF기인적중조선병독감염C17.2신경간세포,Western-blotting분석VEGF단백적표체,병용류식세포술측정세포조망솔적변화.Hoechst33342염색형광현미경관찰조망소체.결과전염pAdCMVVEGF165적C17.2신경간세포성공표체VEGF단백;결양후신경간세포조망솔위(19.98±0.55)%,이전염pAdCMV VEGF165후적세포조망솔위(10.38±0.48)%(P<0.01),전염pAdCMV VEGF165+VEGF반의과핵탈양핵산경간세포조망솔위(19.07±0.64)%,여대조조무현저차이(P>0.05).결론선병독개도적VEGF165기인능고효표체VEGF;외원성VEGF대C17.2신경간세포구유항조망작용,능구제고C17.2신경간세포대결양적내수성,유리우신경간세포적생존.
Objective To study the effects of vascular endothelial growth factor (VEGF) gene transfer on hypoxia-induced apoptosis of neural stem cells in vitro. Methods C17.2 neural stem cells cultured in vitro were infected by recombinant adenovirus containing VEGF gene and cultured under hypoxic condition.VEGF expression in these cells was detected by Western blotting, and the apoptotic index was calculated from results of triphosphate-biotin nick end-labeling (TUNEL) assay.Flow cytometry was employed to examine the changes in the cell apoptotic rate after VEGF gene transfer, and the apoptotic bodies were observed under fluorescence microscope with Hoechst33342 staining. Results The expression of VEGF was significantly increased in pAdCMV VEGF165-infected cells, resulting in inhibition of the apoptosis of C 17.2 neural stem cells induced by hypoxia manifested by a significantly lower apoptotic rate of the stem cells transfected by pAdCMV VEGF165 than that of the untransfected cells (10.38%±0.48% vs 19.98 %±0.55%, P<0.01) and of thecellstransfected with pAdCMV VEGF165 along with VEGF anti-sense oligodeoxynucleotide (19.07%±0.64%,P<0.01) after hypoxia. Conclusions Recombinant adenovirus can efficiently mediate VEGF gene transfer into C17.2 neural stem cells, resulting in high expression of the exogenous VEGF in vitro, which effectively reduces C 17.2 neural stem cell apoptosis induced by hypoxia.