Lipofectamine介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞

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胞嘧啶脱氨酶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:细胞学基因水平体外实验,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,体质量18-20 g,用于骨髓间充质干细胞的分离培养.连接产物转化感受态大肠杆菌DH5a由大连理工大学干细胞与组织工程研发中心提供.Lipofectamine~(TM)2000脂质体为Invitrogen产品.方法:取连接产物转化感受态大肠杆菌DH5a,提取质粒DNA,对质粒plRES2-cGFP1-CD进行Xhol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Lipofectamine~(TM)2000介导法转染第3代鼠骨髓间充质干细胞.主要观察指标:重组质粒的鉴定,流式细胞仪检测鼠骨髓间充质干细胞表面标记表达,荧光倒置显微镜观察细胞转染36,48 h后胞嘧啶脱氨酶基因的表达.结果:质粒plRES2-AcGFP1-CD酶切产物经琼脂糖凝胶电泳后,于1.0～1.5 kb处有1条带出现,符合胞嘧啶脱氨酶基因长度.流式细胞仪检测显示细胞表面标记CD45呈阴性,CD44,CD90,CD105呈阳性.plRES2-AcGFP1-CD基因转染36 h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表达,48 h后细胞仍有荧光表达,且强度明显增强.结论:脂质体介导的胞嘧啶脱氨酶基因在鼠骨髓间充质干细胞中成功表达,于转染48 h后达峰值.

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

BACKGROUND: The particular bystander effect of suicide gene can remarkably kill tumor cells. Meanwhile, 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 plRES2-AcGFP1-CD mediated by liposome transfected into bone marrow mesenchymal stem cells (BMSCs) and its gene expression.DESIGN, TIME AND SETTING: A cytologic experiment at genetic level was performed at Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology from May to December 2007.MATERIALS: A total of 6 C57BL mice of SPF degree and weighing 18-20 g were used for separation and culture of BMSCs.Escherichia coli DH5a was provided by Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology. Lipofectamine~(TM) 2000 was provided by Invitrogen, China.METHODS: The DNA plasmids were extracted from transformed Escherichia coli DH5a. plRES2-AcGFP1-CD plasmid was identified by BamHI/Xhol double digestion. The BMSCs derived 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 LipofectamineTM 2000 mediation.MAIN OUTCOME MEASURES: Identification of recombinant plasmids; the expressions of surface markers on BMSCs were detected by flow cytometry; the expressions of cytosine deaminase gene were observed at 36 and 48 hours after transfection under fluorescent inverted microscope.RESULTS: After agarose gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of plRES2-AcGFP1-CD plasmids, and the band was accorded with the length of cytosine deaminase gene in the length. Flow cytometry demonstrated that the cells were negative for CD45 but positive for CD44, CD90 and CD105. Fluorescent inverted phase contrast microscope suggested that at 36 hours after plRES2-AcGFP1-CD gene transfection an expression of green fluorescent protein was found in the BMSCs; additionally, at 48 hours after transfection, the expression of green fluorescent protein remained and the intensity was remarkably increased.CONCLUSION: The liposome-mediated plRES2-AcGFP1-CD gene successfully expressed in BMSCs, and the expression peaked at 48 hours after transfection.