逆转录病毒载体介导IL 6基因转导成纤维细胞的表达

目的将ＩＬ６基因转导至成纤维细胞ＮＩＨ３Ｔ３，并使转染株有效地表达ＩＬ６，为ＩＬ６转基因治疗奠定基础．方法利用重组载体构建技术将质粒ｐＵＣＩＬ６ｃＤＮＡ的目的片段连接于逆转录病毒载体上，并以脂质体介导的方法将重组载体转染包装细胞ＰＡ３１７，以Ｇ４１８筛选克隆细胞，浓缩克隆细胞上清以制备重组病毒液，继之感染ＮＩＨ３Ｔ３细胞后，进行Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析，检测目的基因在靶细胞的整合与转录水平．结果成功地构建了重组载体ｐＺＩＰＩＬ６ｃＤＮＡ，筛选出抗生较强的克隆细胞，制备了高滴度的重组病毒液．杂交结果表明转导株３Ｔ３ＩＬ６具有ＩＬ６基因的整合和相应ｍＲＮＡ的高表达．结论ＩＬ６基因能稳定整合至靶细胞并进行有效的转录表达，为ＩＬ６基因治疗的应用奠定了可靠的基础

IL 6 expression in transferred NIH3T3 cells by retrovirus vector *

AIM To study the transfer and expression of interleukin 6 gene in NIH3T3 cells for IL 6 gene therapy. METHODS Human IL 6 gene was reconstructed in retrovirus vector and transferred into incasing cells PA317 by lipofectamine mediated method. The clones of the cells transferred with hIL 6 were selected by G418. Targeted NIH3T3 cells were infected with the virus granules secreted from PA317 and also selected by G418. The insertion and expression of hIL 6 gene in NIH3T3 cells were analysed with Southern blot and Northern blot. RESULTS Human IL 6 retrovirus vector (pZIPIL 6) was successfully reconstructed. Positive clones which strongly resisted G418, were selected and hIL 6 retrovirus granules with high biological activity were prepared. Southern blot and Northern blot results confirmed the stable transfer and high expression of hIL 6 in NIH3T3 cells. CONCLUSION Human IL 6 can be successfully transferred and effectively expressed in targeted cells by retroviral vector and may be used in gene therapy.