發(fā)布時(shí)間：2019-02-12 13:22

【摘要】：目的：探索人的小涎腺中成纖維樣細(xì)胞的體外分離、培養(yǎng)方法,并通過分析其體外增殖和多向分化能力,結(jié)合免疫表型檢測,鑒定其干細(xì)胞性質(zhì)。并進(jìn)一步探索小涎腺中成纖維樣單克隆細(xì)胞的體外培養(yǎng)、擴(kuò)增方法,并鑒定其性質(zhì)。 方法：組織塊貼壁培養(yǎng)法原代分離、培養(yǎng)人小涎腺成纖維樣細(xì)胞；觀察原代及傳代后不同代數(shù)細(xì)胞形態(tài)的變化；通過繪制MTT細(xì)胞生長曲線、計(jì)算克隆形成率和觀察不同代數(shù)細(xì)胞擴(kuò)增速度來分析細(xì)胞的增殖能力；直接免疫熒光法流式細(xì)胞學(xué)檢測第三代細(xì)胞的免疫表型；將細(xì)胞向中胚層起源的成骨、脂肪和軟骨細(xì)胞,內(nèi)胚層起源的肝細(xì)胞和外胚層起源的神經(jīng)元細(xì)胞誘導(dǎo)分化,鑒定其多項(xiàng)分化潛能；96孔板稀釋鋪板法進(jìn)行成纖維樣單克隆細(xì)胞的培養(yǎng),并進(jìn)行成骨,成脂肪和成軟骨誘導(dǎo)分化。 結(jié)果：成功的在體外分離培養(yǎng)出人小涎腺成纖維樣細(xì)胞,傳至第四代細(xì)胞形態(tài)保持良好。細(xì)胞體外增殖能力旺盛。流式檢測結(jié)果證實(shí)細(xì)胞表達(dá)普遍認(rèn)可的間充質(zhì)干細(xì)胞標(biāo)記CD13、CD29、CD44、CD90、CD73、CD105、CD166,不表達(dá)造血干/祖細(xì)胞標(biāo)記CD117、CD34、CD45。成脂肪誘導(dǎo)一周左右均有脂滴形成,oil red 0染色陽性,并逐漸增多。成骨誘導(dǎo)8天后,堿性磷酸酶表達(dá)陽性率100%,19天后骨鈣素大量表達(dá),并形成鈣結(jié)節(jié)。成軟骨誘導(dǎo)第8天有極少量細(xì)胞Alcian Blue染色陽性,誘導(dǎo)19天后Alcian Blue染色和Collagen II免疫細(xì)胞化學(xué)染色均為陽性。誘導(dǎo)第24天,RT-PCR檢測各分化方向的特異性基因：脂肪細(xì)胞aP2、C/EBPa. PPARy,成骨細(xì)胞osteopontin,軟骨細(xì)胞collagen II、aggrecan基因均為陽性表達(dá)。向肝細(xì)胞誘導(dǎo)14天,形成肝樣細(xì)胞,并表達(dá)AFP、ALB和CK18。向神經(jīng)元誘導(dǎo)5h后,細(xì)胞表現(xiàn)為神經(jīng)元樣形態(tài),nestin陽性細(xì)胞增多。成纖維樣單克隆細(xì)胞有較強(qiáng)的多項(xiàng)分化能力。 結(jié)論：所建立的體外分離培養(yǎng)體系能夠獲得大量人小涎腺成纖維樣細(xì)胞,體外增殖能力強(qiáng),免疫表型類似于間充質(zhì)干細(xì)胞,在誘導(dǎo)培養(yǎng)條件下能分化為中胚層起源的成骨,脂肪和軟骨細(xì)胞,內(nèi)胚層起源的肝細(xì)胞和外胚層起源的神經(jīng)干細(xì)胞。證明此細(xì)胞為小涎腺間充質(zhì)干細(xì)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：2420473