血管内皮细胞生长因子反义寡核苷酸抑制脑动静脉畸形血管内皮细胞的增殖(英文)

Inhibitory effect of vascular endothelial growth factor-antisense oligonucleotide on the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation

背景:采取反义基因治疗技术控制血管内皮细胞生长因子基因表达,遏止血管生成,是脑血管外科中治疗人脑动静脉畸形的崭新课题。目的:观察血管内皮细胞生长因子反义寡核苷酸对人脑动静脉畸形血管内皮细胞增殖的抑制作用。设计:观察对比实验。单位:解放军沈阳军区总医院神经外科。材料:实验于2006-08/2006-12在解放军沈阳军区总医院的全军神经医学研究所完成。收集2006年解放军沈阳军区总医院神经外科18例脑动静脉畸形患者手术切除的完整人脑动静脉畸形新鲜标本。男12例,女6例;平均40岁。脑动静脉畸形按Spetzler分级:Ⅱ级10例,Ⅲ级8例。全部病例术前均经全脑血管造影证实。人脑动静脉畸形标本获取术前经患者或其家属知情并签同意书。内皮细胞生长添加剂(ECGS;美国Sigma),391型DNA自动合成仪(上海生工利用美国PE公司),厌氧培养箱(浙江产DY-1型),人血管内皮细胞生长因子酶联检测试剂盒购于北京TBD公司,进口分装,96E酶标仪(ERMA,INC)。细胞周期分析试剂盒(BD公司),流式细胞仪(FACSCalibur,BD公司)。方法:①实验过程:采用组织块贴壁法培养人脑动静脉畸形血管内皮细胞,实验用传至3代细胞,随机分成反义组、正义组和对照组,每组4瓶细胞。反义组、正义组分别采用人工合成血管内皮细胞生长因子正义、反义硫代脱氧寡核苷酸,经阳性脂质体包裹后转染体外培养的人脑动静脉畸形血管内皮细胞,对照组不予处理,将各组细胞置于37℃、体积分数0.95N2、0.05CO2厌氧培养箱分别孵育2,4和8h。②实验评估:测定细胞周期。测定细胞血管内皮细胞生长因子蛋白含量。检测细胞血管内皮细胞生长因子mRNA表达。主要观察指标:各组细胞缺氧不同时间点血管内皮细胞生长因子mRNA和蛋白表达及细胞增殖指数。结果:①血管内皮细胞生长因子mRNA表达:对照组细胞缺氧2,4,8h后血管内皮细胞生长因子mRNA水平高于缺氧前(P<0.05),反义组缺氧2,4,8h后血管内皮细胞生长因子mRNA水平低于对照组(P<0.05)。②血管内皮细胞生长因子蛋白含量:对照组缺氧2,4,8h后血管内皮细胞生长因子蛋白含量高于缺氧前(P<0.05),反义组缺氧2,4,8h后血管内皮细胞生长因子蛋白低于对照组(P<0.05)。③细胞增殖指数:对照组人脑动静脉畸形内皮细胞缺氧4,8h后细胞增殖指数(P<0.05)。反义组缺氧4,8h后低于对照组(P<0.05)。结论:缺氧可能在基因转录水平诱导血管内皮细胞生长因子表达,反义血管内皮细胞生长因子能够显著抑制缺氧诱导的人脑动静脉畸形内皮细胞血管内皮细胞生长因子基因表达和细胞增殖。

BACKGROUND：Antisense gene therapy offers immense promise for the management of human cerebral arteriovenous malformation through inhibiting expression of vascular endothelial growth factor and angiogenesis in endothelial cells. OBJECTIVE： To observe the inhibitory effect of vascular endothelial growth factor-antisense oligonucleotide （VEGF-ASODN） on the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation.DESIGN： Observational contrast study. SETTING： Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA. MATERIALS： The experiment was carried out in the Neuromedical Institute, General Hospital of Shenyang Military Area Command of Chinese PLA from August to December 2006. A total of 18 patients with human cerebral arteriovenous malformation were selected from Department of Neurosurgery, Shenyang General Hospital of Military Area Command of Chinese PLA. There were 12 males and 6 females and their mean age was 40 years. Cerebral arteriovenous malformation was classified based on Spetzler grade： grade Ⅱ （n =10） and grade Ⅲ （n=8）. All cases were diagnosed with whole cerebral angiography before operation and they provided the confirmed consent. Main reagents were detailed as follows： endothelial cell growth supplements （ECGS, Sigma, USA）, 391 DNA automatic synthetic device （Shanghai Shenggong Liyong Company, PE, USA）, anaerobic incubator （DY-1, Zhejiang）, human vascular endothelial growth factor enzyme-linked kit （TBD Company, Beijing）, 96E enzyme-labeling device （ERMA, INC）, cell cycle analytical reagent kit （BD Company）, and flow cytometer （FACS Calibur, BD Company）. METHODS： ①Experimental procedure： Tissue explants adherent method was used to culture vascular endothelial cells from human cerebral arteriovenous malformation. The third generated cells were used and randomly divided into antisense group, sense group and control group with four bottles of cells in each group. Sense and antisense phosphorothioate oligodeoxynucleotides of artificial vascular endothelial growth factor selected from the antisense group and the sense group were covered with positive liposomes, and then they were used to transfected vascular endothelial cells cultured from human cerebral arteriovenous malformation; however, cells in the control group were not dealt with any treatments. Cells in the three groups were incubated in anaerobic incubator （including 0.95 volume fraction of N2 and 0.05 volume fraction of CO2） at 37 ℃ for 2, 4 and 8 hours, respectively. ② Experimental evaluation： Cell cycles were measured, protein content of vascular endothelial growth factor was measured, and mRNA expression of vascular endothelial growth factor was also detected. MAIN OUTCOME MEASURES： Expression of mRNA and protein of vascular endothelial growth factor and proliferation exponent at different times of hypoxia. RESULTS： ①mRNA expression of vascular endothelial growth factor： At 2, 4 and 8 hours after hypoxia, mRNA expression of vascular endothelial growth factor was higher than that before hypoxia in the control group （P 〈 0.05）;however, mRNA expression was lower in the antisense group than that in the control group （P 〈 0.05）. ② Protein content of vascular endothelial growth factor： At 2, 4 and 8 hours after hypoxia, protein content of vascular endothelial growth factor was higher than that before hypoxia in the control group （P 〈 0.05）; however, protein content was lower in the antisense group than that in the control group （P 〈 0.05）. ③ Proliferation exponent： At 4 and 8 hours after hypoxia,proliferation exponent of endothelial cells cultured from human cerebral arteriovenous malformation was higher than that before hypoxia in the control group （P 〈 0.05）; however, proliferation exponent was lower in the antisense group than that in the control group （P 〈 0.05）. CONCLUSION： Hypoxia may induce gene expression of vascular endothelial growth factor in endothelial cells at the transcriptional level. Antisense vascular endothelial growth factor can obviously inhibit gene expression of vascular endothelial growth factor cultured from human cerebral arteriovenous malformation and proliferation under hypoxic conditions.