分离鉴定人小涎腺间充质干细胞

发布时间：2019-02-12 13:22

【摘要】：目的：探索人的小涎腺中成纤维样细胞的体外分离、培养方法,并通过分析其体外增殖和多向分化能力,结合免疫表型检测,鉴定其干细胞性质。并进一步探索小涎腺中成纤维样单克隆细胞的体外培养、扩增方法,并鉴定其性质。方法：组织块贴壁培养法原代分离、培养人小涎腺成纤维样细胞；观察原代及传代后不同代数细胞形态的变化；通过绘制MTT细胞生长曲线、计算克隆形成率和观察不同代数细胞扩增速度来分析细胞的增殖能力；直接免疫荧光法流式细胞学检测第三代细胞的免疫表型；将细胞向中胚层起源的成骨、脂肪和软骨细胞,内胚层起源的肝细胞和外胚层起源的神经元细胞诱导分化,鉴定其多项分化潜能；96孔板稀释铺板法进行成纤维样单克隆细胞的培养,并进行成骨,成脂肪和成软骨诱导分化。结果：成功的在体外分离培养出人小涎腺成纤维样细胞,传至第四代细胞形态保持良好。细胞体外增殖能力旺盛。流式检测结果证实细胞表达普遍认可的间充质干细胞标记CD13、CD29、CD44、CD90、CD73、CD105、CD166,不表达造血干/祖细胞标记CD117、CD34、CD45。成脂肪诱导一周左右均有脂滴形成,oil red 0染色阳性,并逐渐增多。成骨诱导8天后,碱性磷酸酶表达阳性率100%,19天后骨钙素大量表达,并形成钙结节。成软骨诱导第8天有极少量细胞Alcian Blue染色阳性,诱导19天后Alcian Blue染色和Collagen II免疫细胞化学染色均为阳性。诱导第24天,RT-PCR检测各分化方向的特异性基因：脂肪细胞aP2、C/EBPa. PPARy,成骨细胞osteopontin,软骨细胞collagen II、aggrecan基因均为阳性表达。向肝细胞诱导14天,形成肝样细胞,并表达AFP、ALB和CK18。向神经元诱导5h后,细胞表现为神经元样形态,nestin阳性细胞增多。成纤维样单克隆细胞有较强的多项分化能力。结论：所建立的体外分离培养体系能够获得大量人小涎腺成纤维样细胞,体外增殖能力强,免疫表型类似于间充质干细胞,在诱导培养条件下能分化为中胚层起源的成骨,脂肪和软骨细胞,内胚层起源的肝细胞和外胚层起源的神经干细胞。证明此细胞为小涎腺间充质干细胞。
[Abstract]:Aim: to explore the method of isolation and culture of fibroblasts from human salivary glands in vitro, and to identify the stem cell properties by analyzing their ability of proliferation and multidirectional differentiation in vitro and combining with immunophenotypic detection. The methods of culture, amplification and identification of fibroblast-like monoclonal cells in small salivary glands in vitro were further explored. Methods: human salivary gland fibroblasts were isolated and cultured by tissue mass adherent culture method, and the morphological changes of different algebraic cells were observed after primary culture and passage. By drawing the growth curve of MTT cells, calculating the clone forming rate and observing the expansion rate of different algebraic cells, the proliferative ability of the cells was analyzed, and the immunophenotype of the third generation cells was detected by direct immunofluorescence flow cytology. The cells were induced to differentiate into mesoderm-derived osteoblasts, adipose and chondrocytes, hepatocytes derived from endoderm and neuron cells from ectoderm. The culture of fibroblast-like Monoclonal cells was carried out by 96 hole plate dilution and paving method, and osteogenesis, adipogenic and chondrogenic differentiation were carried out. Results: human small salivary gland fibroblasts were successfully isolated and cultured in vitro. The ability of cell proliferation in vitro is exuberant. Flow cytometry confirmed that the cells expressed universally recognized mesenchymal stem cell labeled CD13,CD29,CD44,CD90,CD73,CD105,CD166, did not express hematopoietic stem / progenitor cell marker CD117,CD34,CD45. Lipid droplets were positive for, oil red 0 staining after one week of adipogenic induction, and increased gradually. After 8 days of osteogenesis, the positive rate of alkaline phosphatase expression was 100% and 19 days later, osteocalcin was highly expressed and calcium nodules were formed. On the 8th day after chondrogenic induction, very few cells were positive for Alcian Blue staining. After 19 days of induction, Alcian Blue staining and Collagen II immunocytochemical staining were both positive. On the 24th day of induction, RT-PCR was used to detect the specific genes in different differentiation directions: adipocyte aP2,C/EBPa.. Collagen II,aggrecan gene expression in osteopontin, chondrocytes of PPARy, osteoblasts was positive. Hepatocytes were induced for 14 days to form hepatocyte-like cells and expressed AFP,ALB and CK18.. After induction to neurons for 5 h, the cells showed neuron-like morphology and the number of nestin positive cells increased. Fibroblast-like monoclonal cells have strong differentiation ability. Conclusion: a large number of human small salivary gland fibroblasts can be obtained by the established culture system in vitro. The immunophenotype is similar to that of mesenchymal stem cells, and can differentiate into mesoderm-derived osteogenesis. Fat and chondrocytes, endoderm derived hepatocytes and ectodermal neural stem cells. It is proved that this cell is a small salivary gland mesenchymal stem cell.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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