摘要
目的构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础。方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化。体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达。结果成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达。结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad-mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞。
Objective To construct adenovirus vectors contained milL-18 and observation the expression in rBMSCs and explore experiment study for gene therapy of glioma. Methods mIL-18 gene was subcloned into the shuttle plasmid pAdtrackCMV, then linearized the vector and convert competent cell BJ5183 which contain the adenovirus frame plasmid pAdEasy-1. The homologous recombination plasmid was packaged by HEK293 cells and purified by CsCI. The cell im- munophenotype was detected by flow cytometry. The expression of mIL-18 was measured by fluorescence microscope, RT-PCR and ELISA. Results The adenovirus vector Ad- mIL-18 was successfully constructed, which was labeled by green fluorescent protein （GFP） and effectively expressed in rBMSCs. Conclusion pAdEasy-1 is an effective gene trans- fection system, rBMSCs transfected with Ad- mIL-18 can serve as the seeds cells for the gene therapy of glioma.
出处
《西部医学》
2012年第6期1067-1070,共4页
Medical Journal of West China
