含绿色荧光蛋白报告基因的TIMP-2真核表达载体的构建及其在人成釉细胞瘤中的表达

目的构建含有绿色荧光蛋白报告基因（GFP）的TIMP-2真核表达载体PcDNA3．1（＋）／GFP-TIMP-2，并探讨其在成釉细胞瘤（AB）中的表达情况。方法应用RT-PCR技术从体外培养的人AB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AB细胞。流式细胞仪测定转染效率，倒置相差荧光显微镜观察绿色荧光，RT-PCR检测转染前后TIMP-2mRNA的表达量的改变。结果构建的PcDNA3．1（＋）／GFP-TIMP-2经酶切和测序鉴定证明和预期结果一致。PcDNA3．1（＋）-TIMP-2转染人AM细胞后TIMP-2mRNA的表达量增加。结论成功构建TIMP-2真核表达载体PcDNA3．1（＋）／GFP-TIMP-2并转染至人AB细胞。

The construction and expression of PcDNA3.1(+)/GFP-TIMP-2 in human ameloblastoma cell

OBJECTIVE: 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, molecmicrolar 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 Flow 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 mRNA 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.