论文部分内容阅读
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18～20g,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。LipofectamineTM 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LipofectamineTM 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36,48h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1.0～1.5kb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48h后达峰值。
BACKGROUND: The bystander effect of suicide gene can significantly provide tumor cell killing effect, and it is also used in combination with radiotherapy and immunotherapy, and overcomes the defect of low gene transduction efficiency. Cytosine deaminase can produce a powerful bystander effect. OBJECTIVE: To observe the effect and gene expression of liposome-mediated eukaryotic expression vector cytosine deaminase transfected mouse bone marrow mesenchymal stem cells. DESIGN, TIME AND SETTING: The level of cytology genes in vitro was performed at R & D Center of Stem Cell and Tissue Engineering, Dalian University of Technology on May 12, 2007. MATERIALS: Six C57BL mice with SPF grade and 18 ~ 20g body weight were used for the isolation and culture of bone marrow mesenchymal stem cells. The ligation product was transformed into competent E. coli DH5a and provided by R & D Center for Stem Cell and Tissue Engineering of Dalian University of Technology. LipofectamineTM 2000 liposomes are Invitrogen products. METHODS: The ligation product was transformed into E. coli DH5a. The plasmid DNA was extracted and subjected to XhoI and BamHI double digestion of the plasmid plRES2-cGFP1-CD for transfection. Bone marrow mesenchymal stem cells (MSCs) were isolated and purified from the bilateral lower extremities of femur and tibia of mice. The cells were passaged to the third generation to make single cell suspensions. Fluorescent labeled CD44, CD45, CD90 and CD105 antibodies were added, and LipofectamineTM 2000 The third generation of murine bone marrow mesenchymal stem cells were transfected by mediation. MAIN OUTCOME MEASURES: The identification of recombinant plasmids, the expression of the surface markers of mouse bone marrow mesenchymal stem cells were detected by flow cytometry. The cytosine deaminase gene expression was observed by fluorescence inverted microscope 36 and 48 hours after transfection. Results: The band of plasmid p1RES2-AcGFP1-CD digested with agarose gel electrophoresis showed a band of 1.0 ~ 1.5kb, which was in accordance with the length of cytosine deaminase gene. Flow cytometry showed that cell surface markers CD45 negative, CD44, CD90, CD105 positive. After transfection of plRES2-AcGFP1-CD gene for 36h, the expression of green fluorescent protein in mouse bone marrow mesenchymal stem cells was observed under fluorescence inverted phase contrast microscope. After 48h, the cells still had the fluorescence expression, and the intensity was significantly enhanced. Conclusion: The liposome-mediated cytosine deaminase gene was successfully expressed in murine bone marrow-derived mesenchymal stem cells and reached its peak 48h after transfection.