Wang Yan, Qiao Hui, Peng Ting-Sheng, Li Yang, Zhang Meng, Liang Hui-Zhen, Qiu Ju-Shi
Department of Pathology, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510089, China.
Zhonghua Bing Li Xue Za Zhi. 2007 Mar;36(3):190-5.
To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.
Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 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.
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 concentration-dependent 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 G0 approximately G1 phase, apoptosis and necrosis.
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 double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.
探讨缺氧条件下血管内皮生长因子（VEGF）在骨肉瘤细胞系中的表达情况以及单纯疱疹病毒I型胸苷激酶/丙氧鸟苷（HSV1-TK/GCV）系统对转染骨肉瘤细胞的靶向杀伤作用。
构建含缺氧反应元件（HRE）启动子的真核表达质粒，以表达反义VEGF165 cDNA及潮霉素磷酸转移酶-胸苷激酶（HyTK）融合基因。然后用脂质体介导的基因转移方法将重组载体转染至骨肉瘤细胞系MG63。采用聚合酶链反应（PCR）及逆转录-聚合酶链反应（RT-PCR）证实TK基因的存在及表达。通过噻唑蓝（MTT）比色法及混合共培养实验检测转染细胞在常氧或缺氧条件下对GCV的敏感性及HSV1-TK/GCV系统的“旁观者效应（BSE）”。采用酶联免疫吸附测定（ELISA）法检测缺氧条件下VEGF蛋白的表达。通过流式细胞术检测细胞周期分布。此外，用电子显微镜记录超微结构改变。
成功构建真核表达载体pBI-HRE-AsVEGF165 -HyTK。通过PCR及RT-PCR证实转基因及其mRNA表达，从而建立了转染细胞系MG63TV。GCV对转染细胞具有浓度依赖性毒性。缺氧条件下对GCV毒性的敏感性比常氧条件下高100倍。混合培养实验表明，缺氧条件下“旁观者效应”显著增强。缺氧条件下转基因细胞的VEGF表达较正常条件下降50%。在缺氧及GCV作用下，MG63TV细胞的DNA合成受到抑制，同时处于G0~G1期的细胞增多，出现凋亡及坏死。
由HRE启动子驱动的反义VEGF表达联合缺氧可靶向抑制人骨肉瘤细胞中的VEGF表达，增强HSV-TK/GCV系统的选择性杀伤作用及旁观者效应。本研究中的双基因共表达系统为骨肉瘤的同步抗血管生成及自杀基因治疗方法提供了实验依据。
