摘要
目的构建牙本质基质蛋白1(DMP1)绿色荧光融合蛋白pEGFP-DMP1,确定该重组质粒转染猪口腔粘膜成纤维细胞(POMFs)及骨髓间质干细胞(MSCs)的最佳条件,评价DMP1转基因修饰对细胞生物学特性及DMP1基因表达的影响。方法构建pEGFP-DMP1真核表达载体,使用不同参数评价脂质体介导基因修饰POMFs及MSCs的转染效率,检测转染后细胞的DMP1基因表达及蛋白表达情况。结果成功构建DMP1真核表达载体pEGFP-DMP1,酶切后得到4.7kb和1.5kb的片段,当脂质体用量为5μl、质粒2μg、转染细胞密度为30%时,DMP1基因的表达比例最高,POMFs及MSCs的最佳转染时间分别为3h和45min。转基因48h后的细胞可见有DMP1基因表达,免疫组化染色显示DMP1阳性。结论选择合适的转染条件可使pEGFP-DMP1绿色荧光融合蛋白重组质粒高效转染POMFs及MSCs,并可检测到DMP1基因及DMP1蛋白表达。
Objective To construct recombinant plasmid pEGFP-DMP1 and to evaluate the expression of dental matrix protein l （DMP1） in transfected porcine oral mucosa fibroblast （POMFs） and mesenchymal stem cells （MSCs） and the influence of the transfection.Methods The full length porcine DMP1 cDNA was linked into an eukaryotic expression vector pEGFP-C1. POMFs and MSCs were transfected with the pEGFP-DMP1 and the effect of different parameters on transfection efficiency of livestock cells to express a reporter gene was determined. The expression of DMP1 gene of transfected POMFs and MSCs were detected by RT-PCR. The expression of DMP1 was examined by immunocytochemical staining.Results The constructed pEGFP-DMP1 could produced 4.7kb and 1.5kb fragments. Lipofectamine 5μl obtained greater transfection efficiency with plasmid concentration 2μg/ml reachinig 30% of all cells. The optimized transfection time to express the pEGFP-DMP1 gene of POMFs and MSCs is 3h and 45 minute respectively. DMP1 gene were expressed 48h after transfection. Immunohistochemical staining showed that DMP1 was positive in transfected cells.Conclusion Optimal transfection parmeters can lead to greater transfection efficiency of pEGFP-DMP1 gene by lipofectamine on porcine somatic cells. The transfection of porcine DMP1 in POMEs and MSCs can induce the expression of DMP1 gene and protein.
出处
《现代口腔医学杂志》
CAS
CSCD
北大核心
2007年第4期406-409,共4页
Journal of Modern Stomatology
