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目的:观察逆转录病毒介导的胞嘧啶脱氨酶(E.coli-cytosinedeaminase,CD)基因、单纯疱疹病毒胸苷激酶(herpessimplexvirus-thymidinekinase,HSV-tk)基因转染肝门部胆管癌的作用。方法:在脂质体lipofectamine的介导下将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,收集其病毒上清,转染FRH细胞,用RT-PCR检测双自杀基因的表达。给予前体药物5-氟胞嘧啶(5-flourocytosine,5-Fc)和(或)无环鸟苷(ganciciovir,GCV)后,用四甲基偶氮唑蓝比色(MTT)法测定转基因组及未转基因组FRH细胞存活率。在0.8cm裸鼠肝门部胆管癌模型转染入双自杀基因后,给予5-Fc500mg/kg体重,GCV100mg/kg体重,1次/d,共10d,观察肿瘤生长情况。结果:双自杀基因在FRH细胞中可稳定表达,联合使用5-Fc和GCV对靶细胞增殖的杀伤作用及旁杀伤效应高于单独使用5-Fc或GCV。在裸鼠实验性肝门部胆管癌,实验组肿瘤的生长明显受到抑制(P<0.05);双自杀基因与单自杀基因的作用有显著差异(P<0.05)。结论:逆转录病毒介导自杀基因可有效地杀死肝门部胆管癌细胞,明显抑制裸鼠肝门部胆管癌生长,双自杀基因共表达较单一自杀基因有更强的抗肿瘤作用。
OBJECTIVE: To observe the effect of retrovirus-mediated transfection of hepatocellular cholangiocarcinoma with cytosine deaminase (CD) gene and herpes simplex virus-thymidine kinase (HSV-tk) gene . Methods: The retroviral vector PWZLneoCDglytk containing double suicide genes was introduced into packaging cell PA317 by lipofectamine. The virus supernatant was collected and transfected into FRH cells. The expression of double suicide gene was detected by RT-PCR. After administration of the prodrug 5-flourocytosine (5-Fc) and / or ganciciovir (GCV), the trans genome was determined by MTT assay And the non-transgenic group FRH cell survival rate. After transfection into double suicide gene in nude mouse model of hilar cholangiocarcinoma, the mice were given 5-Fc500mg / kg body weight, GCV100mg / kg body weight, once / d for 10 days to observe the tumor growth. Results: The double suicide gene was stably expressed in FRH cells. The combined effect of 5-Fc and GCV on target cell proliferation and para-killing was higher than that of 5-Fc or GCV alone. The growth of tumor in experimental group was significantly inhibited (P <0.05) in nude mouse experimental hilar cholangiocarcinoma. The effect of double suicide gene and single suicide gene was significantly different (P <0.05). CONCLUSION: The retrovirus-mediated suicide gene can effectively kill hilar cholangiocarcinoma cells, inhibit the growth of hilar cholangiocarcinoma in nude mice, and double suicide gene co-expression has a stronger anti-tumor effect than single suicide gene.