人小涎腺成纤维样单克隆细胞成骨分化.doc

人小涎腺成纤维样单克隆细胞成骨分化[摘要]目的:探索人小涎腺成纤维样单克隆细胞的体外培养、扩增方法,鉴定其性质,研究向成骨细胞分化的潜能。方法:组织块贴壁法原代培养人小涎腺细胞,收集成纤维样细胞,用96孔板稀释铺板法克隆培养,免疫荧光和RT-PCR检测细胞表型,并进行成骨诱导分化,通过骨钙素、一型胶原免疫细胞化学染色及碱性磷酸酶、茜素红钙结节染色鉴定成骨分化。结果:成功的在体外扩增培养出人小涎腺成纤维样单克隆细胞,免疫荧光证实细胞表达CD13、CD29、CD106和CD44,RT-PCR显示CK18、α-SMA、vimentin、CD106、nestin基因表达与人骨髓间充质干细胞相似。成骨诱导8天后,碱性磷酸酶表达阳性率100%,19天后骨钙素和一型胶原大量表达,并形成钙结节。结论:所建立的分离培养体系能够获得大量人小涎腺成纤维样单克隆细胞,免疫表型类似于间充质干细胞。在成骨诱导培养条件下具有良好的向成骨细胞分化的潜能。 [关键词]小涎腺;单克隆培养;成骨诱导;组织工程骨 [中图分类号]Q813.1 [文献标识码]A [文章编号]1008-6455(2010)04-0523-04 Osteogenic differentiation of human small salivary gland fibroblast-like monoclonal cells DU Ming-juan, ZHAO Zhen-min, YAN Li, YIN Ning-bei, SONG Tao, LI Hai-dong (Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Peking 100144, China) Abstract:ObjectiveExplore the method of culturing and proliferating human small salivary gland fibroblast-like monoclonal cells in vitro, identify the character of the cells and study the potential of osteoblast differentiation.MethodsHuman small salivary gland cells was primary cultured using tissue adherent method, we collect the fibroblast-like cells and clonal culture them using 96-well plates dilution decking method, and detect cell phenotype by immunofluorescence and RT-PCR. Then we start osteogenic inducing, and identify osteogenic differentiation through osteocalcin, collagen typeⅠimmunocytochemistry staining and alkaline phosphatase(AKP), alizarin red staining.ResultsWe successfully amplify human small salivary gland fibroblast-like monoclonal cells in vitro, these cells express CD13, CD29, CD106 and CD44 proved by immunofluorescence, and express gene CK18, α-SMA, vimentin, CD106, nestin revealed by RT-PCR similar to human bone marrow-derived mesenchymal stem cells. After 8 days of osteogenic differentiation, the positive expression rate of AKP is 100%, after 19 days the expressive number of osteocalcin and collagen typeⅠis large, and there are many calcium nodules formation.C