脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞

Transfection of mouse bone marrow mesenchymal stem cells with liposome mediated cytosine deaminase genes

背景：自杀基因独有的旁观者效应,可显著提供肿瘤细胞杀伤效果,同时还与放射治疗、免疫基因治疗联合应用,并克服了基因转导效率低的缺陷。胞嘧啶脱氨酶（cytosinedeaminase,CD）即可产生强大的旁观者效应。目的：观察脂质体介导真核表达载体CD基因转染鼠骨髓间充质干细胞的效果及其基因表达。设计、时间及地点：细胞学基因水平实验,于2007-05/12在大连理工大学干细胞与组织工程研发中心完成。材料：SPF级C57BL纯系小鼠6只,由大连医科大学实验动物中心提供。连接产物转化感受态大肠杆菌DH5α由大连理工大学干细胞与组织工程研发中心提供。LipofectamineTM2000脂质体为Invitrogen产品。方法：取连接产物转化感受态大肠杆菌DH5α,提取质粒DNA,对质粒pIRES2-AcGFP1-CD进行XhoI和BamHI双酶切,用于转染。取小鼠双侧下肢股骨和胫骨,贴壁法分离纯化骨髓间充质干细胞,传至第3代制成单细胞悬液,加入荧光标记的CD44,CD45,CD90,CD105抗体后,采用Lipofectamine2000介导法转染第3代骨髓间充质干细胞。主要观察指标：重组质粒的鉴定,流式细胞仪检测骨髓间充质干细胞表面标记表达,细胞转染后CD基因的表达。结果：质粒pIRES2-AcGFP1-CD酶切产物经琼脂糖凝胶电泳后,于1.0-1.5kb处有一条带出现,符合CD基因长度。细胞表面标记CD45呈阴性,CD44,CD90,CD105呈阳性。pIRES2-AcGFP1-CD基因转染36h后,荧光倒置相差显微镜下可见小鼠骨髓间充质干细胞有绿色荧光蛋白的表达,48h后细胞仍有荧光表达,且强度明显增强。结论：脂质体介导的CD基因在鼠骨髓间充质干细胞中成功表达,于转染48h后达峰值。

BACKGROUND： The particular bystander effect of suicide gene can remarkably kill tumor cells. It can be used together with radiotherapy as well as immune gene therapy, and overcome the defect of low gene transduction efficiency. Cytosine deaminase （CD） can generate a powerful bystander effect. OBJECTIVE： To observe the effect of a eukaryotic expression plasmid pIRES2-AcGFP1-CD mediated by liposome transfected into bone marrow mesenchymal stem cells （BMSCs） and its gene expression. DESIGN, TIME AND SETTING： A cytologic experiment of genetic level was performed at Research and Development Center of Stem Cell and Tissue Engineer, Dalian University of Technology from May to December 2007. MATERIALS： A total of six C57BL mice of SPF degree were provided by Experimental Animal Center of Dalian Medical University. E. DH5a was provided by Research and Development Center of Stem Cell and Tissue Engineer, Dalian University of Technology. LipofectamineTM 2000 was the product of Invitrogen, China. METHODS： The DNA plasmids were extracted from transformed into the competent E. DH5a. plRES2-AcGFP1-CD plasmid was identified by BamHI/Xhol double digestion. The BMSCs from mouse femur and tibia were cultured and purified by adhesion method in vitro. Signal cell suspension prepared by BMSCs of the third generation was cultured by adding fluorescence-labeled CD44, CD45, CD90 and CD105 antibodies. BMSCs of the third generation were transfected by lipofectamine 2000 mediation. MAIN OUTCOME MEASURES： Identification of recombinant plasmids. The expressions of surface markers on BMSCs were detected by fluoroscopy. The expressions of CD gene were observed after transfection. RESULTS： After agarose gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of plRES2-AcGFP1-CD plasmids and accorded with the length of CD gene in the length. The cells were negative for CD45, and positive for CD44, CD90, and CD105. Under the fluorescent inverted phase contrast microscope, the expressions of green fluorescent protein in the BMSCs were found at 36 hours after transfection. At 48 hours after transfection, the expressions of green fluorescent protein remained and the intensity increased obviously. CONCLUSION： The expression of CD gene mediated by liposome in BMSCs is successful, and it reaches the peak 48 hours after transfection.