pIRES2-EGFP-hBMP_2载体的构建及对人牙龈成纤维细胞表型的影响  

Construction of pIRES2-EGFP-hBMP2 and its effect on the phenotype of gingival fibroblasts

作　　者：唐昊喆[1] 王勤涛[1] 董广英[1] 马志伟[1] 丁敖[1] 李长春[2] 孙迎春[2] 王莉[3] 

机构地区：[1]第四军医大学口腔医学院,陕西西安710033 [2]天津市塘沽区口腔医院颌面外科,天津300450 [3]第四军医大学基础部,陕西西安710033

出　　处：《第四军医大学学报》2007年第16期1497-1500,共4页Journal of the Fourth Military Medical University

摘　　要：目的:构建hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2并观察其对人牙龈成纤维细胞表型的影响.方法:采用RT-PCR方法从人成骨肉瘤细胞中克隆hBMP2基因,连接T载体并测序正确后与真核表达载体pIRES2-EGFP连接,酶切鉴定后转染入HEK293细胞中,利用荧光显微镜和WesternBlot进行表达鉴定,最后转染人牙龈成纤维细胞,通过MTT实验观察转染hBMP2后对人牙龈成纤维细胞增殖特性、ALP活性、OC含量、体外矿化能力的影响.结果:成功克隆了人BMP2基因,酶切鉴定及测序均正确,真核表达载体pIRES2-EGFP-hBMP2能正确表达hBMP2蛋白,hBMP2对人牙龈成纤维细胞增殖特性无影响,但可增强其ALP活性、OC含量及体外矿化能力.结论:构建了hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2,该基因可促进人牙龈成纤维细胞向成骨细胞方向分化.AIM： To construct and identify the eukaryotic expression vector pIRES2-EGFP-hBMP2 which expressing human hBMP2 and to observe its effects on the phenotype of human gingival fibroblast. METHODS： hMBP2 gene was cloned from human osteogenic sarcoma by RT-PCR and inserted into T-vector. After DNA sequencing verification, it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP . After identification by double digestion, the pIRES2-EGFP-hBMP2 was transfected into HEK293 cells to verify the expression by fluorescence microscope and Western Blot. The construct was transfected into human gingival fibroblast to detect the effects of hBMP2 on the proliferation, ALP activity, content of OC and the ex vivo mineralization capacity of fibroblast by MTY. RESULTS： Human BMP2 gene was cloned successfully and verified by double digestion and DNA sequencing. The constructed eukaryotic expression vector pIRES2- EGFP-hBMP2 expressed hBMP2 correctly, hBMP2 did not affect the proliferation of human gingival fibroblast but increased the ALP activity, OC content and ex vivo mineralization capacity. CONCLUSION： The eukaryotic expression vector pIRES2- EGFP-hBMP2 expressing hBMP2 is constructed successfully and this gene promotes human gingival fibroblast to differentiate toward osteoblast.

关 键 词：牙龈 成纤维细胞 骨形态发生蛋白质2 转染 成骨细胞 

分 类 号：R780.2[医药卫生—口腔医学]