缺氧反应元件启动子-反义血管内皮生长因子_(165)cDNA和单纯疱疹病毒1-胸苷激酶基因共表达治疗骨肉瘤的体外研究  

Suppression of osteosarcoma in vitro bv coexnression of antisense VEGF_165 cDNA and thvmidine kinase gene

作　　者：王燕[1] 乔慧[1] 彭挺生[1] 李扬[1] 张萌[1] 梁惠珍[1] 丘钜世[1] 

机构地区：[1]中山大学中山医学院病理学教研室

出　　处：《中华病理学杂志》2007年第3期190-195,共6页Chinese Journal of Pathology

基　　金：国家自然科学基金(30300354)

摘　　要：目的探讨缺氧条件下,转基因细胞血管内皮生长因子(VEGF)表达变化以及单纯疱疹病毒1-胸苷激酶(HSV1-TK)/丙氧鸟苷(GCV)系统对转基因细胞的特异杀伤作用。方法以缺氧诱导因子-1/缺氧反应元件(HIF-1/HRE)基因调节系统为载体,采用 DNA 重组技术构建由 HRE 启动子驱动的含人反义 VEGF_(165) cDNA 全长和 HSV1-TK基因的真核表达质粒,将其稳定转染人骨肉瘤细胞系 MG63。应用 PCR 和 RT-PCR 方法检测 TK 基因的整合及转录;用 MTF 法和混合共培养实验分别检测转基因细胞在缺氧或不缺氧条件下对GCV 的敏感性以及 HSV1-TK/GCV 系统的"旁观者效应";ELISA 法检测细胞在缺氧条件下 VEGF 表达变化;流式细胞仪检测细胞周期改变及电镜观察细胞超微结构变化。结果成功构建了由 HRE 启动子驱动的含反义 VEGF_(165) cDNA 全长和潮霉素磷酸转移酶-单纯疱疹病毒胸腺嘧啶激酶(HyTK)基因的真核表达质粒 pBI-HRE-AsVEGF_(165)-HyTK;得到转基因细胞系 MG63TV;检测到 MG63TV 细胞内 TK 基因的 DNA 和 mRNA。缺氧条件下,转基因细胞存活率与 GCV 浓度呈现剂量依赖性,对 GCV 的敏感性提高了100倍;HSV1-TK/GCV 系统旁观者效应明显增强;肿瘤细胞 VEGF 分泌下降50%。缺氧和 GCV 共同作用使转基因细胞 DNA 合成抑制,细胞周期阻滞于 G_0～G_1期,细胞发生凋亡和坏死。结论利用肿瘤缺氧,HRE 启动子能够实现反义VEGF_(165)对体外培养的肿瘤细胞 VEGF 表达的靶向性抑制作用,并能增强 HSV1-TK/GCV 系统对肿瘤细胞的特异性杀伤作用和旁观者效应。该双基因共表达载体系统为实践联合抗血管生成和自杀基因治疗骨肉瘤提供了实验依据。Objective To investigate the effect of VEGF expression in osteosareoma cell line and the target killing effect of HSV1-TK/C, CV system on transfected osteosarcoma cells under hypoxia conditions. Methods Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF,~ cDNA and Hygromycin phospho- transferase-thymidine kinase （HyTK） fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MC,63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and ＂bystander effect （BSE）＂ of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations. Results The eukaryotic expression vector pBI-HRE-AsVEGF165-HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentrationdependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the ＂bystander effect＂ was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at Go 165 GI phase, apoptosis and necrosis. Conclusions Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The don

关 键 词：骨肉瘤 细胞 培养的 缺氧诱导因子 血管内皮生长因子类 

分 类 号：R738.1[医药卫生—肿瘤]