肿瘤坏死因子肝癌特异性重组载体的构建及其介导的肝癌特异性基因治疗的研究

Construction of retroviral vectors to induce strong hepatoma cell-specific expression of murine tumor necrosis factor gene and the hepatoma-specifie gene therapy mediated by the recombinant retroviral vectors.

将小鼠肿瘤坏死因子(mTNF)cDNA克隆到逆转录病毒载体MNSM构建MNSM-SV40-mTNF,用小鼠白蛋白增强子/启动子Alb e/p元件置换MNSM-SV40-mTNF中SV40启动子构建成MNSM-Alb e/p-mTNF。构建物经脂质体转染法引入包装细胞PA137中制备重组病毒,在Polybrene存在条件下感染小鼠肿瘤细胞,经RNA和TNF活性分析证明Alb e/p可驱动TNF基因在合成白蛋白的小鼠肝癌细胞中特异转录和表达,转TNFα基因小鼠肝癌细胞体内致瘤性消失,体内特异抑制亲代肝癌生长。重组病毒及产病毒PA317于肝癌瘤体内显著抑制肝癌生长,特异地延长荷肝癌小鼠存活期。病理及免疫组化分析证实in situ基因治疗后,肝癌广泛坏死、出血并伴有大量Mac-11^+、CD_8^+和CD_(25)^+炎细胞浸润和纤维化。

Murine tumor necrosis factor(mTNF)cDNA was inserted into the polylinker site of MNSM retroviral vector to create pMNSM-SV40-mTNF. Albumin enhancer/promoter (Alb e/p) sequence was used to replace SV40 early region promoter of pMNSM-SV40-mTNF vector to create pMNSM-Alb e/p-mTNF recombinant retroviral vector. The retroviral constructs were introduced into amphotropic retroviral packaging cells PA317. Production of the recombinant retroviruses was accomplished by the lipofectamine-mediated gene transfer procedure. The murine tumor cells were infected with the retroviruses in the presence of polybrene. Dot hybridization of total RNA from modified tumor cell with mTNF cDNA probe and mTNF bioassay demonstrated that transcription and expression of mTNF gene drived by Alb e/p were markedly high in the hepatoma cells which produced albumin, and inhibited in the non-hepatoma tumor cells. The hepatoma cells modified with mTNF gene lost its tumorgenicity and significantly inhibited the growth of the parental hepatoma in vivo. High-titer MNSM-Alb e/p-mTNF retroviruses or the high-titer retroviruses producing packaging cells, after intra-tumoral injection, specifcally inhibited the growth of the hepatoma, and significantly prolonged the survival period of the hepatoma-loaded mice. Biopsy and immunohistochemical assay of the hepatoma during in vivo gene therapy, showed the occurrence of extensive tumor necrosis, bleeding, and CD8+, CD25+, CD4+ lymphocytic infiltration and fibrosis.