摘要
目的阻断可诱导共刺激途径协同骨髓间充质干细胞(mesenchymal stem cells,MSCs)可能能增强急性移植物抗宿主病(acute graft versus host disease,aGVHD)的防治作用。文中旨在构建携带小鼠可诱导共刺激分子(inducible costimula-tor,ICOS)基因的重组腺病毒载体,转染小鼠骨髓MSCs,为转基因治疗提供实验基础。方法设计含有EcoRⅠ、SalⅠ酶切位点的引物,PCR扩增ICOS,将扩增产物克隆到穿梭质粒PDC318上,筛选阳性克隆,测序鉴定正确,将其与含有5型腺病毒右臂的质粒pPE3-F11B-EGFP在293细胞中同源重组,筛选获得含有ICOS基因的重组腺病毒质粒,行PCR鉴定。重组腺病毒质粒转染293细胞,扩增后用氯化铯密度梯度离心纯化病毒,用50%组织培养感染剂量法测定病毒滴度,并转染小鼠MSCs。结果重组腺病毒质粒经鉴定,证实含有ICOS基因,病毒滴度为6.54×109pfu/ml。转染小鼠MSCs,荧光显微镜下可见较多绿色荧光蛋白表达,流式细胞仪检测ICOS表达率为90.16%。结论成功构建了携带小鼠ICOS基因的重组腺病毒载体,并获得高滴度的病毒,能高效转染小鼠MSCs,为后续研究奠定了基础。
Objective Blockade of the inducible costimulator （ICOS） and its ligand signal pathway combined with mesenchymal stem cells （MSCs） may enhance the preventive ability of acute graft against host disease. This study was to construct a recombinant adenovirus vector carrying the mouse ICOS gene and transfect mouse MSCs so as to provide an experimental basis for transgene therapy. Methods The amplification products of ICOS by PCR with a pair of primers containing EcoR I and Sal I restriction endonuclease sites were subcloned into the shuttle plasmid PDC318. The plasmid PDC318-mICOS was co-transfected with plasmid pPE3- F11B-EGFP containing the right arm of adenovirus 5 into 293 cells to produce a new recombinant adenovirus, followed by PCR identification. The adenovirus was packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. The viral titer was determined by the method of 50% tissue culture infective dose （TCIDSO）. Results The results of PCR assay indicated that the target gene was successfully inserted into the recombinant adenovirus vector. The viral titer was 6.54× 10^9pfu/ml. Fluorescence microscopy showed many high-titer viruses in the transfected MSCs, and the expression of ICOS was 90.16%. Conclusion The recombinant adenovirus vector containing mouse ICOS was successfully constructed and high-titer viruses were obtained, and the vector could efficiently transfect mouse MSCs.
出处
《医学研究生学报》
CAS
北大核心
2012年第9期909-914,共6页
Journal of Medical Postgraduates
基金
国家自然科学基金(30871100)
作者简介
王利平（医学博士）
通讯作者：王健民，E—mail：jmwang@medmail．com．cn
