逆转录病毒载体介导的IL-6大肠癌基因治疗实验研究

Retroviral vector mediated IL6 gene therapy for human colon cancer cells.

目的：探讨ＩＬ６转基因表达对大肠癌细胞的体内外抑制作用。方法：参照分子克隆技术将重组逆转录病毒载体ｐＺＩＰＩＬ６ｃＤＮＡ转染ＰＡ３１７包装细胞，以Ｇ４１８筛选抗药性细胞，常规制备重组病毒液并感染大肠癌ＨＴ２９细胞，采用ＮｏｒｔｈｅｒｎＢｌｏｔ分析基因转录水平，ＥＬＩＳＡ法和ＭＴＴ显色法检测蛋白表达的量与细胞活性，以细胞生长曲线和集落形成实验以及裸鼠移植瘤实验，观察转导株的体内外抑瘤作用。结果：制备了高滴度的重组病毒液（５１×１０５ｃｆｕ／ｍｌ），建立了稳定表达ＩＬ６的转导细胞（ＨＴ２９／ＩＬ６），表达的量与活性分别为１１３２５ｐｇ／ｍｌ与１５０Ｕ／ｍｌ，转导株的细胞群体倍增时间为２５天，对数生长期在４～７天之间，集落形成率和抑制率分别为２２１％和５０％，接种裸鼠皮下的出瘤时间为１３５天，最终瘤体直径在６５～８５ｍｍ之间，以上参数与非转导株ＨＴ２９细胞相比，均有显著差异。结论：通过逆转录病毒载体的介导，ＩＬ６基因能稳定整合在靶细胞染色体，并进行有效的转录表达，ＩＬ６转基因表达可明显抑制大肠癌细胞的体外增殖和体内移植瘤的形成与发展。

Objective: To investigate the regulatory effect of IL6 exression on colon cancer cells. Methods: Human IL6 gene was successfuly reconstructed into retroviral vector pZIPIL6cDNA and transfected into package cells PA317,which were then selected by antibiotic G418Human colon cancer cells HT29 were infected with the prepared recombinant virus granules.IL6 gene were analyzed with Northern Blot at transcription level and the expression of protein were measured with ELISA and MTT method.Furthermore,the tumor repression function of the transfected cells were observed in vivo and in vitro through cells growth curve,plating efficiency and the inoculated tumors in nude mice. Results:High titer recombinant virus granules were prepared and stably expressive transfected cells HT29IL6 were constructed,the amount of the secreted protein was 11325 pg/ml and the activity was 150U/ml.Doubling time of the transfected cells was 25 days and log phase of growth was 47 days,plating efficiency and repression rate were 221%and 50%respectively.The tumor forming time in the inoculated nude mice was 135days and the final diameters of the tumor were among 65 to 85 mm,signifiantly lower than the nontransfected cells. Conclusion: Mediated by retroviral vector,IL6 gene can be stably expressed in the human colon cancer cells,and the expression clearly repress the proliferation of colon cancer cells in vitro and growth of the inocculated tumor in vivo.