【摘要】 目的:利用脂肪酸去饱和酶基因fat-1改变细胞膜脂肪酸组成,进行大肠癌的基因治疗研究。方法:将fat-1基因插入腺病毒载体中,与骨架载体同源重组,构建腺病毒重组载体（Ad-GFP-fat1）,通过包装细胞系（293）产生的腺病毒,感染人大肠癌株HT-29细胞。提取细胞的总RNA,以fat-1基因的反义mRNA作探针,用Northern Blot检测fat-1基因在HT-29细胞内的表达。以流式细胞仪对HT-29细胞G₀/G₁期、S期、G₂/M期所占比例进行检测,分析fat-1基因对HT-29细胞增殖和凋亡的影响。以气相色谱分析仪分析fat-1基因对HT-29细胞细胞膜n-6 PUFAs和n-3 PUFAs含量及n-6/n-3PUFAs比例的影响。将HT-29细胞皮下接种于裸鼠右前肢腋下,建立裸鼠HT-29大肠癌细胞皮下移植瘤模型。成瘤后进行治疗实验,经连续5次治疗,于最后一次治疗后第3天处死小鼠,取肿瘤称重。分析fat-1基因裸鼠体内抗肿瘤效果。结果:通过基因重组技术,得到高滴度的含fat-1基因的重组病毒;腺病毒介导的fat-1基因能够在HT-29细胞中有效表达;fat-1基因的表达可降低HT-29细胞膜n-6/n-3PUFAs的比例,有效抑制HT-29细胞增殖,促进细胞凋亡并能抑制裸鼠移植瘤的发展。结论:fat-1基因的表达,可抑制HT-29细胞的体内外增殖并诱导细胞凋亡,在大肠癌基因治疗中可能具有良好利用价值。更多还原

【Abstract】 Obective: To explore the effect of the fat-1 gene encoding n-3 fatty acid desaturase on the proliferation and apoptosis of human colon cancer cell line HT-29 and to explore its value in the gene therapy for colorectal cancer. Methods: The fat-1 gene was cloned into adenovirus shuttle vector pAd-CMV and then recombinated with backbone vector to construct a recombinant adenoviral vector pAd.GFP.fatl. The vector was transfected into 293 cells to get the recombinant virus infected human colon cancer cell line HT-29. Total RNA of the cells was analyzed by Northern blot. The effect of fat-1 gene on the proliferation and apoptosis of the infected cells was analyzed by flow cytometry. The content of n-6PUFAs/n-3PUFAs was analyzed by Gas Chromatography. The anti-cancer effect of fat-1 gene was studied on HT-29 xenografts in nude mice in vivo. Results: The high titer recombinant virus was obtained. Fat-1 gene can be highly expressed in human colon cancer cell line HT-29. Compared with that of the control cells (Ad.GFP), proliferation of HT-29 cells was inhibited by fat-1 gene. Fat-1 gene can lower the ratio of n-6/n-3PUFAs. The growth of tumors in nude mice is also inhibited by fat-1 gene. Conclusion: Fat-1 gene is of great value in the gene therapy for colorectal cancer.更多还原