中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
32期
6517-6520
,共4页
血管内皮生长因子%脑动静脉畸形%内皮细胞%增殖%基因治疗
血管內皮生長因子%腦動靜脈畸形%內皮細胞%增殖%基因治療
혈관내피생장인자%뇌동정맥기형%내피세포%증식%기인치료
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(FACS Calibur,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0.95 N2、0.05 CO2厌氧培养箱分别孵育2,4和8 h.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2,4,8 h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2,4,8 h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2,4,8 h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2,4,8 h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4,8 h后细胞增殖指数(P＜0.05).反义组缺氧4,8 h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.
揹景:採取反義基因治療技術控製血管內皮細胞生長因子基因錶達,遏止血管生成,是腦血管外科中治療人腦動靜脈畸形的嶄新課題.目的:觀察血管內皮細胞生長因子反義寡覈苷痠對人腦動靜脈畸形血管內皮細胞增殖的抑製作用.設計:觀察對比實驗.單位:解放軍瀋暘軍區總醫院神經外科.材料:實驗于2006-08/2006-12在解放軍瀋暘軍區總醫院的全軍神經醫學研究所完成.收集2006年解放軍瀋暘軍區總醫院神經外科18例腦動靜脈畸形患者手術切除的完整人腦動靜脈畸形新鮮標本.男12例,女6例;平均40歲.腦動靜脈畸形按Spetzler分級:Ⅱ級10例,Ⅲ級8例.全部病例術前均經全腦血管造影證實.人腦動靜脈畸形標本穫取術前經患者或其傢屬知情併籤同意書.內皮細胞生長添加劑(ECGS;美國Sigma),391型DNA自動閤成儀(上海生工利用美國PE公司),厭氧培養箱(浙江產DY-1型),人血管內皮細胞生長因子酶聯檢測試劑盒購于北京TBD公司,進口分裝,96E酶標儀(ERMA,INC).細胞週期分析試劑盒(BD公司),流式細胞儀(FACS Calibur,BD公司).方法:①實驗過程:採用組織塊貼壁法培養人腦動靜脈畸形血管內皮細胞,實驗用傳至3代細胞,隨機分成反義組、正義組和對照組,每組4瓶細胞.反義組、正義組分彆採用人工閤成血管內皮細胞生長因子正義、反義硫代脫氧寡覈苷痠,經暘性脂質體包裹後轉染體外培養的人腦動靜脈畸形血管內皮細胞,對照組不予處理,將各組細胞置于37 ℃、體積分數0.95 N2、0.05 CO2厭氧培養箱分彆孵育2,4和8 h.②實驗評估:測定細胞週期.測定細胞血管內皮細胞生長因子蛋白含量.檢測細胞血管內皮細胞生長因子mRNA錶達.主要觀察指標:各組細胞缺氧不同時間點血管內皮細胞生長因子mRNA和蛋白錶達及細胞增殖指數.結果:①血管內皮細胞生長因子mRNA錶達:對照組細胞缺氧2,4,8 h後血管內皮細胞生長因子mRNA水平高于缺氧前(P＜0.05),反義組缺氧2,4,8 h後血管內皮細胞生長因子mRNA水平低于對照組(P＜0.05).②血管內皮細胞生長因子蛋白含量:對照組缺氧2,4,8 h後血管內皮細胞生長因子蛋白含量高于缺氧前(P＜0.05),反義組缺氧2,4,8 h後血管內皮細胞生長因子蛋白低于對照組(P＜0.05).③細胞增殖指數:對照組人腦動靜脈畸形內皮細胞缺氧4,8 h後細胞增殖指數(P＜0.05).反義組缺氧4,8 h後低于對照組(P＜0.05).結論:缺氧可能在基因轉錄水平誘導血管內皮細胞生長因子錶達,反義血管內皮細胞生長因子能夠顯著抑製缺氧誘導的人腦動靜脈畸形內皮細胞血管內皮細胞生長因子基因錶達和細胞增殖.
배경:채취반의기인치료기술공제혈관내피세포생장인자기인표체,알지혈관생성,시뇌혈관외과중치료인뇌동정맥기형적참신과제.목적:관찰혈관내피세포생장인자반의과핵감산대인뇌동정맥기형혈관내피세포증식적억제작용.설계:관찰대비실험.단위:해방군침양군구총의원신경외과.재료:실험우2006-08/2006-12재해방군침양군구총의원적전군신경의학연구소완성.수집2006년해방군침양군구총의원신경외과18례뇌동정맥기형환자수술절제적완정인뇌동정맥기형신선표본.남12례,녀6례;평균40세.뇌동정맥기형안Spetzler분급:Ⅱ급10례,Ⅲ급8례.전부병례술전균경전뇌혈관조영증실.인뇌동정맥기형표본획취술전경환자혹기가속지정병첨동의서.내피세포생장첨가제(ECGS;미국Sigma),391형DNA자동합성의(상해생공이용미국PE공사),염양배양상(절강산DY-1형),인혈관내피세포생장인자매련검측시제합구우북경TBD공사,진구분장,96E매표의(ERMA,INC).세포주기분석시제합(BD공사),류식세포의(FACS Calibur,BD공사).방법:①실험과정:채용조직괴첩벽법배양인뇌동정맥기형혈관내피세포,실험용전지3대세포,수궤분성반의조、정의조화대조조,매조4병세포.반의조、정의조분별채용인공합성혈관내피세포생장인자정의、반의류대탈양과핵감산,경양성지질체포과후전염체외배양적인뇌동정맥기형혈관내피세포,대조조불여처리,장각조세포치우37 ℃、체적분수0.95 N2、0.05 CO2염양배양상분별부육2,4화8 h.②실험평고:측정세포주기.측정세포혈관내피세포생장인자단백함량.검측세포혈관내피세포생장인자mRNA표체.주요관찰지표:각조세포결양불동시간점혈관내피세포생장인자mRNA화단백표체급세포증식지수.결과:①혈관내피세포생장인자mRNA표체:대조조세포결양2,4,8 h후혈관내피세포생장인자mRNA수평고우결양전(P＜0.05),반의조결양2,4,8 h후혈관내피세포생장인자mRNA수평저우대조조(P＜0.05).②혈관내피세포생장인자단백함량:대조조결양2,4,8 h후혈관내피세포생장인자단백함량고우결양전(P＜0.05),반의조결양2,4,8 h후혈관내피세포생장인자단백저우대조조(P＜0.05).③세포증식지수:대조조인뇌동정맥기형내피세포결양4,8 h후세포증식지수(P＜0.05).반의조결양4,8 h후저우대조조(P＜0.05).결론:결양가능재기인전록수평유도혈관내피세포생장인자표체,반의혈관내피세포생장인자능구현저억제결양유도적인뇌동정맥기형내피세포혈관내피세포생장인자기인표체화세포증식.
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.
