人反义VEGF基因表达载体的构建及其对卵巢癌细胞增殖的影响  

Construction of antisense VEGF eukaryotic expression vector and effect to ovarian cancer cell

作　　者：严瑞兰[1] 金明[2] 钱新华[3] 惠宏襄[2] 辛晓燕[1] 王建[1] 王德堂[1] 

机构地区：[1]第四军医大学西京医院妇产科,陕西西安710032 [2]第四军医大学分子生物学教研室,陕西西安710032 [3]第一军医大学南方医院儿科,广东广州510060

出　　处：《中国癌症杂志》2001年第4期289-293,共5页China Oncology

基　　金：国家自然科学基金资助 ( 3970 0 14 7)

摘　　要：目的 :构建人反义VEGF基因真核表达载体 ,进行抗血管生成治疗卵巢癌实验。方法 :RT PCR法获得人VEGF基因 ,将VEGF基因反向克隆入真核表达载体pcDNA3中 ,酶切鉴定结果。用此真核表达载体转染人卵巢癌细胞HO 8910 ,G418筛选获得阳性克隆 ,RNA斑点杂交鉴定HO 8910细胞中VEGF表达。Westernblot及间接免疫荧光法检测转染前后HO 8910细胞中VEGF蛋白表达。检测转染前后瘤细胞的生物学性状及裸鼠体内致瘤性。结果 :RT PCR法得到VEGF基因 ,并获得反向构建的VEGF真核表达载体。VEGF反义RNA部分阻断了HO 8910细胞中VEGF表达 ,转染后单个细胞的克隆形成能力明显减弱 ;细胞周期中 ,G1期细胞增多 ,S期细胞减少 ,细胞增殖能力降低 ;形态学超微结构显示细胞器扩张和肿胀 ,核染色质边集 ,凝集成块 ,可见明显的凋亡改变。裸鼠体内致瘤性降低。结论 :成功构建了反义VEGF基因真核表达载体 ,该载体可明显抑制卵巢癌细胞增殖 。Purpose:To constract and identify eukaryotic expression vector carrying human VEGF cDNA to cure human ovarian cancer with antiangiogenesis method. Methods:Reverse transcription PCR for VEGF, VEGF was inserted into eukaryotic exprssion vector pcDNA3 to construct pcDNA3-VEGF(-), in which restriction enzyme analysis was used to confirm the reverse orientation fo the VEGF cDNA from the individual transformants. The vector was transfected into human ovarian cancer cell HO-8910 and the positive clone was screened by G418,VEGF expression was confirmed by RNA dot blot. The VEGF expression of HO-8910 cells before or after transfection was detected by Western blot and imunofluorescence.The biological characteristics of HO-8910 cells before and after transfectionwas inspected. Results: VEGF gene was obtained by RT-PCR, the pcDNA3-VEGF(-) vector was obtained. VEGF expression was blocked by anisense VEGF RNA. The clone formation ability of singal cell was decreased after transfected, G 1 phase cells were increased and S Phase celIs were decreased in cell cycle. The proliferation of HO-89l0 cell was reduced. The formation rate and growth speed of xengrafted tumor slowed down. Conclusions:The successful construction of antisense VEGF eukaryotic expression vector is of significance for ovarian cancer-specific antisense gene therapy.

关 键 词：血管内皮生长因子 真核表达载体 构建 卵巢癌 细胞增殖 VEGF基因 

分 类 号：R737.31[医药卫生—肿瘤]