白介素-18基因修饰人脑胶质瘤U87/hIL-18细胞的建立  

Establishment of human glioma U87/hIL-18 cells genetically modified by interleukin-18 gene

作　　者：戴学军[1] 袁振华 林健[1] 徐敏敏 赵刚[1] 王建业[1] 王伟民[1] 

机构地区：[1]中国人民解放军广州军区广州总医院神经外科,广东广州510010 [2]本元正阳基因技术股份有限公司,北京100176

出　　处：《中国微侵袭神经外科杂志》2009年第1期29-32,共4页Chinese Journal of Minimally Invasive Neurosurgery

基　　金：广东省自然科学基金(编号:001122);中国博士后科学基金面上资助项目(编号:20070420766)

摘　　要：目的构建人白介素18(hIL-18)基因真核表达载体质粒,建立hIL-18基因修饰并呈稳定表达的人脑胶质瘤U87/hIL-18细胞。方法从人淋巴细胞cDNA文库钓取hIL-18基因后行PCR扩增,利用pcDNA3.1(+)质粒为载体,构建hIL-18真核表达质粒pcDNA3.1/hIL-18。将构建的重组质粒转染人胶质瘤细胞U87,采用G418筛选单克隆细胞株,ELISA检测培养上清中的hIL-18蛋白含量,提取细胞RNA,RT-PCR方法检测hIL-18基因在U87/hIL-18细胞中的表达。结果所得hIL-18cDNA序列与Gene Bank比对一致,构建的质粒经PCR、酶切及测序鉴定正确。质粒转染U87细胞后,经加压筛选获得hIL-18基因稳定、高效表达的U87/hIL-18单克隆细胞株,细胞培养72h的上清中,hIL-18蛋白含量达131.2pg/ml,且RT-PCR结果显示细胞中hIL-18基因表达呈阳性。结论成功构建hIL-18真核表达质粒pcDNA3.1/hIL-18,并建立U87/hIL-18单克隆细胞株,为hIL-18基因修饰的胶质瘤疫苗研究打下了基础。Objective To construct a eukaryotic expression plasmid carrying human interleukin-18 （hIL-18） gene and establish human glioma U87/hIL-18 cells modified by hIL-18 with stable expression. Methods Human IL-18 gene was amplified from human lymphocyte cDNA library using PCR and inserted into plasmid pcDNA3.1 （＋） to consmtct the eukaryotic expression plasmid pcDNA3.1/hIL-18. Human glioma U87 cells were transfected with the constructed plasmid and screened for monoclonal cell strains by G418. The hIL-18 in supematant was examined and RNA extracted by ELISA test, and the expression of hIL-18 gene in U87/hIL-18 cell was detected by ELISA and RT-PCR. Results The sequence of hIL-18 was consistent with Gene Bank and the constructed plasmid was confirmed by PCR, restriction enzyme digestion and sequencing. A monoclonal cell line with stably and highly expressed hIL-18 gene was gained by pressurized screening after U87 cells were transfected, and the protein level ofhIL-18 in 72-hour culture supematant was 131.2 pg/ml. RT-PCR demonstrated positive hlL-18 gene expression in U87/hIL-18 cells. Conclusion The eukaryotic expression plasmid pcDNA3.1/hIL-18 has been successfully constructed and the U87/hIL-18 strain was established, which provides a foundation for developing glioma vaccine modified by IL-18 gene.

关 键 词：神经胶质瘤 白细胞介素18 质粒 U87细胞 

分 类 号：R739.4[医药卫生—肿瘤]