碱性成纤维细胞生长因子基因在骨膜成骨细胞中的表达

Expression of basic fibroblast growth factor gene in periosteal osteoblasts

目的:探讨碱性成纤维细胞生长因子基因在骨膜.成骨细胞中的表达情况,以为其基因转移在骨组织工程中的应用奠定基础。方法:实验于2003-09在广州军区总医院医学实验科完成。采用聚乙烯亚胺介导重组真核表达载体pcDNA3.1-碱性成纤维细胞生长因子经转染至原代培养的成骨细胞中,培养36h后,采用细胞免疫组化、免疫印迹杂交、酶联免疫吸附法、四甲基偶氮唑盐微量酶反应比色等方法检测碱性成纤维细胞生长因子在成骨细胞中的表达情况以及其生物学活性。结果:①pcDNA3.1-碱性成纤维细胞生长因子转染的成骨细胞能分泌表达碱性成纤维细胞生长因子至细胞培养液中,表达量约为(1.56±0.08)ng/mL,高于未经转染的细胞培养液[(0.53±0.048)ng/ml,(P<0.01)]。②经转染的成骨细胞的培养上清可以明显促进3T3细胞的增殖,表明成骨细胞所分泌表达的碱性成纤维细胞生长因子具有一定的生物学活性。结论:碱性成纤维细胞生长因子基因能够转移至骨膜成骨细胞中,经转染的细胞能够超表达碱性成纤维细胞生长因子。

AIM： To investigate the expression of basic fibroblast growth factor （bFGF） gene in periosteal osteoblasts in order to lay a foundation of the application of bFGF gene transfer in bone tissue engineering. METHODS： The experiment was carried out in the Department of Medical Laboratory, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA in September 2003. Polyethylenimide （jetPEITM） mediated pcDNA3.1-bFGF recombinant vector was transferred into primarily cultured osteoblasts. After 36 hours, the expression of bFGF and its biological activities were detected with immunohistochemisty, Western blot, enzyme-linked immunoadsordent assay （ELISA） and methylthiazol-tetrazolium （MTT） colorimetry. RESULTS： The pcDNA3.1-bFGF transfected osteoblasts could express bFGF protein into the cellular culture medium, the concentration was higher than that in the non-transfected one [（1.56±0.08）, （0.53±0.048） ng/mL, P 〈 0.01]. ② The supernatant of transfected osteoblasts could obviously accelerate the proliferation of 3T3 cells,which indicated that the osteoblasts secreted and expressed bFGF had certain biological activities. CONCLUSION： The bFGF gene can transfer into periosteal osteoblasts, and the transfected osteoblasts can overexpress bFGF.