pIRES2-AcGFP1-CD真核表达载体构建及其在骨髓间充质干细胞中的表达

Construction of pIRES2-AcGFP1-CD eukaryotic expression plasmid and its expression in bone marrow mesenchymal stem cells

背景：骨髓间充质干细胞在体外易分离和扩增，还易于外源基因的转入及表达，在人类医学上被认为是一种理想的治疗性细胞和基因治疗中的靶细胞。目的：探讨脂质体介导胞嘧啶脱氨酶基因转染兔骨髓间充质干细胞及其基因的表达。设计：单一样本观察。单位：大连市干细胞与组织工程研发中心，大连医科大学基础医学院生化室。材料：实验于2006—03／2007—06在大连市干细胞与组织工程研发中心及大连医科大学基础医学院生化室完成。新西兰大白耳兔，5月龄，雌雄不拘，体质量2．0～2．5kg。方法：以大肠杆菌JM109基因组DNA为模板，用PCR方法获得目的基因片段，定向克隆至载体pMD19-T，限制性内切酶消化鉴定、基因测序后，构建pIRES2-AcGFP1-CD真核表达质粒。同时对兔骨髓间充质干细胞取材、培养、鉴定。真核表达质粒经酶切鉴定后，采用Lipofectamine2000介导法转染经过鉴定的兔骨髓间充质干细胞。并于转染后24h在倒置荧光显微镜下观察绿色荧光蛋白的表达。主要观察指标：表达载体的构建及基因转染骨髓间充质干细胞鉴定。结果：实验克隆出胞嘧啶脱氨酶基因，并将其与带荧光的真核表达载体pIRES2-AcGFP1连接。经转染兔骨髓间充质干细胞24h后，在倒置荧光显微镜488am蓝光激发下观察，pIRES2-AcGFP1-CD组和pIRES2-AcGFP1空载体组均可见细胞发出绿色荧光，未经转染的细胞未见发出绿色荧光，说明胞嘧啶脱氨酶基因成功转染了骨髓间充质干细胞。结论：骨髓间充质干细胞有望成为胞嘧啶脱氨酶基因治疗中的理想载体。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are easily isolated and amplified, and facilitate the exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are ideal therapeutic cells and target cells in the gene therapy. OBJECTIVE： To investigate liposome-mediated cytosine deaminase （CD） gene transfecting rabbit BMSCs and its gene expression. DESIGN： A single sample observation. SETTING： Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University. MATERIALS： This study was performed at in the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were included in this study. METHODS： The CD gene was obtained from E.coli JM109 DNA by polymerase chain reaction （PCR）. The fragment was cloned into pMD19-T vector. After restriction enzyme BamHl/XhoI digestion analysis and DNA sequence analysis, pIRES2-AcGFPI-CD eukaryotic expression plasmid was constructed. Meanwhile, BMSCs were harvested, cultured and identified. After enzyme digestion of eukaryotic expression plasmid, the rabbit BMSCs were transfected by Lipofectamine 2000-mediated method. Twenty-four hours after transfection, expression of green fluorescent protein was observed under an inverted fluorescent microscope. MAIN OUTCOME MEASURES： Construction of eukaryotic expression plasmid and identification of CD gene-transferred BMSCs. RESULTS： CD gene was cloned and connected to eukaryotic expression plasmid with green fluorescence. Twenty-four hours after transfecting rabbit BMSCs, it was found under an inverted microscope that under the excitation of 488 nm blue light, green fluorescence appeared in the plRES2-AcGFPI-CD and pIRES2-AcGFPI empty-plasmid transfected BMSCs, but not in the non-transfected ones. It indicates that CD gene successfully transferred BMSCs. CONCLUSION： BMSCs are ideal vectors in the CD gene therapy.