【摘要】： 目的:本實(shí)驗(yàn)通過體外分離培養(yǎng)同種異體大鼠間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs),了解其一般生物特性及低免疫原性,探討同種異體大鼠MSCs在肝細(xì)胞生長因子(hepatocyte growth factor HGF)和成纖維生長因子4(basic fibroblast factor FGF-4)誘導(dǎo)下向肝樣細(xì)胞(hepatocyte-like cells)橫向分化的可能性,以期為臨床同種異體骨髓間充質(zhì)干細(xì)胞治療終末期肝病提供理論基礎(chǔ)。 方法: 1.無菌條件下取出大鼠的兩側(cè)股骨,沖洗骨髓腔獲得細(xì)胞,采用全骨髓培養(yǎng)法體外培養(yǎng)細(xì)胞,貼壁篩選純化大鼠MSCs并進(jìn)行體外培養(yǎng)、擴(kuò)增。 2.取第三代MSCs進(jìn)行流式細(xì)胞儀測試CD34、CD45、CD29、CD106,并誘導(dǎo)其向脂肪細(xì)胞分化。 3.取兩只大鼠第三代MSCs混合培養(yǎng),加入一定濃度的HGF和FGF-4對(duì)MSCs誘導(dǎo)培養(yǎng),未加入生長因子組作為對(duì)照組,觀察誘導(dǎo)細(xì)胞的形態(tài)學(xué)變化,免疫組化檢測培養(yǎng)誘導(dǎo)細(xì)胞的細(xì)胞角蛋白-18(cytokeratin-18 CK-18)、細(xì)胞角蛋白-19(cytokeratin-19 CK-19)及波形蛋白Vimentin;ELISA等方法檢測甲胎蛋白(alpha-fetoprotein AFP)、白蛋白(albumin ALB)、堿性磷酸酶(alkaline phosphatase ALP)。 結(jié)果: 1.剛分離接種的大鼠骨髓MSCs形態(tài)大小均一。大鼠MSCs在24h內(nèi)貼壁,10天左右達(dá)到對(duì)數(shù)生長期。細(xì)胞傳至第三代時(shí)候,細(xì)胞融合成單層,梭形突起變長,排列有明顯方向性,呈旋渦狀、魚群樣。 2.第三代大鼠MSCs流式細(xì)胞儀檢測CD34、CD45陰性,CD29、CD106陽性,MSC誘導(dǎo)后分化為脂肪細(xì)胞。 3.混合培養(yǎng)的大鼠MSCs經(jīng)HGF和FGF-4共同誘導(dǎo)后7天后,開始觀察到少量細(xì)胞變成短梭形,誘導(dǎo)14天后可以觀察到細(xì)胞變成三角形或是橢圓形,隨時(shí)間的延長橢圓形細(xì)胞越多。而未加誘導(dǎo)因子的對(duì)照組未觀察到三角形及橢圓形細(xì)胞的出現(xiàn),細(xì)胞仍呈梭形生長,并相互融合。當(dāng)誘導(dǎo)的細(xì)胞大部分變成橢圓形后,行免疫組化及ELASA檢測,結(jié)果顯示CK18、CK19、Vimentin表達(dá)陽性,AFP、ALB、ALP出現(xiàn)明顯改變。 結(jié)論: 1. HGF和FGF-4的共同作用可以促進(jìn)同種異體大鼠MSCs橫向分化為肝樣細(xì)胞。 2. 10%胎牛血清的DMEM低糖培養(yǎng)基適合大鼠間充質(zhì)干細(xì)胞培養(yǎng)和分化。 3.骨髓源性間充質(zhì)干細(xì)胞具有低免疫原性。 4.骨髓源性MSCs可成為肝組織工程主要種子細(xì)胞來源。
[Abstract]:Objective: to investigate the general biological characteristics and low immunogenicity of allogeneic rat mesenchymal stem cells (Mesenchymal stem cells,MSCs) isolated and cultured in vitro. To investigate the possibility of transversely differentiation of allogeneic MSCs into hepatoid cells (hepatocyte-like cells) induced by hepatocyte growth factor (hepatocyte growth factor HGF) and fibroblast growth factor (4 (basic fibroblast factor FGF-4). To provide a theoretical basis for the treatment of end-stage liver disease with allogeneic bone marrow mesenchymal stem cells. Methods: 1. The femur of both sides of the rat was removed under aseptic condition, and the cells were obtained by washing the medullary cavity. The cells were cultured in vitro by whole bone marrow culture method. The MSCs of rats was screened and purified by adherent method and cultured and amplified in vitro. 2. The third generation MSCs was used to detect CD34,CD45,CD29,CD106, and induce it to differentiate into adipocytes. 3. The third generation MSCs of two rats were mixed cultured, and the MSCs was induced by adding a certain concentration of HGF and FGF-4. The growth factor group was not added to the control group, and the morphological changes of the induced cells were observed. Immunohistochemical detection of cytokeratin-18 (cytokeratin-18 CK-18), cytokeratin 19 (cytokeratin-19 CK-19) and vimentin Vimentin; in cultured cells Detection of alpha-fetoprotein (alpha-fetoprotein AFP), albumin (albumin ALB), alkaline phosphatase (alkaline phosphatase ALP).) by ELISA and other methods Results: 1. The bone marrow MSCs of the newly isolated and inoculated rats was uniform in size. Rat MSCs adheres to the wall within 24 hours and reaches logarithmic growth stage about 10 days. At the third generation, the cells were fused into monolayer, fusiform protuberances became longer, arranged in obvious directionality, appeared as swirl, fish. 2. The third generation of rat MSCs flow cytometry detected CD34,CD45 negative, CD29,CD106 positive, MSC induced differentiation into adipocytes. 3. After co-induction of MSCs by HGF and FGF-4 for 7 days, a small number of cells were observed to be fusiform. After 14 days of induction, triangular or elliptical cells were observed, and the number of oval cells increased with time. In the control group without induction factor, triangular and oval cells were not observed, and the cells were still fusiform and fused with each other. When most of the induced cells became oval, the positive expression of CK18,CK19,Vimentin and AFP,ALB,ALP were detected by immunohistochemistry and ELASA. Conclusion: 1. The interaction of HGF and FGF-4 can promote the transversely differentiation of MSCs into hepatoid cells in allogeneic rats. 2. DMEM medium with 10% fetal bovine serum was suitable for rat mesenchymal stem cell culture and differentiation. 3. Bone marrow derived mesenchymal stem cells have low immunogenicity. 4. Bone marrow derived MSCs may be the main source of seed cells in liver tissue engineering.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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