【作者】 张磊涛； 黄洪章； 曾东林； 张彬；

【Author】 Zhang Lei-tao, Huang Hong-zhang, Zeng Dong-tin, Zhang Bin Department of Oral Maxillofacial Surgery, the Second Affiliated Hospital, Sun Yat-Sen University

【机构】 中山大学附属第二医院口腔颌面外科；

【摘要】 <正>目的:构建含有绿色荧光蛋白报告基因的TIMP-2真核表达载体PcDNA3．1(+)／GFP-TIMP-2。探讨 TIMP-2基因转染体外培养的人成釉细胞瘤细胞的表达情况。方法:应用RT-PCR技术从体外培养的人成釉细胞瘤细胞中获得TIMP-2目的基因片段,采用分子克隆技术构建该基因的真核表达载体 PcDNA3．1(+)／GFP-TIMP-2并以脂质体为介导转染至体外培养的人成釉细胞瘤细胞。流式细胞仪(FCM) 测定转染效率,倒置相差荧光显微镜观察绿色荧光,RT-PCR检测转染前后TIMP-2 mRNA的表达量的改更多还原

【Abstract】 Objectives: To construct the eukaryotic expression vector of TIMP-2 gene and to explore its expression in human ameloblastoma cell in vitro. Methods: The aimed gene fragment was obtained by RT-PCR. And then, molecular cloning technology and enzyme digestion were used to connect the gene with the plasmid PcDNA3.1(+),which can be expressed in eukaryotic cells and a report gene: green fluorescent protein gene (GFP) was already existed in the plasmid. We named the eukaryotic expression vector, which contended our aimed gene TIMP-2 as well as report gene GFP, PcDNA3.1(+)/GFP-TIMP-2. The vector was identified by PCR analysis, EcoR I and Xho I restriction analysis and Sequence analysis. After the PcDNA3.1(+)/GFP-TIMP-2 was transfected into cultured human ameloblastoma cell, RT-PCR and How Cytometry(FCM) and Microscope wre respectively performed to evaluate the effect of transfection and expression. Results: The constructed vector PcDNA3.1(+)/GFP-TIMP-2 was proved correct by enzyme digestion and sequencing analysis. After PcDNA3.1(+)/GFP-TIMP-2 was trasnfected into cultured human ameloblastoma cell . the rate of transfection is 47.6%(Analysis report of FCM), the green fluorescence was found in plasm (observed with fluo-microwave), the expression of TIMP-2 niRNA was elevated 2.4 times compared with the control group. Conclusions: PcDNA3.1(+)/GFP-TIMP-2 was successfully constructed and it could be transfected into cultured human ameloblastoma cell. It may be benefit to further study of the relationship between the TIMP-2 gene and the behaviour of ameloblastoma.更多还原