本文關(guān)鍵詞：體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向肝系細(xì)胞定向分化實(shí)驗(yàn)研究　出處：《南昌大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 胎肝細(xì)胞 肝細(xì)胞生長(zhǎng)因子 肝細(xì)胞 分化

【摘要】：目的：骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells，MSCs)是一種存在于骨髓內(nèi)的非造血干細(xì)胞，由于其具有自我更新能力及多向分化潛能，在組織器官修復(fù)及再生方面顯示出良好的應(yīng)用前景而倍受研究者關(guān)注。本實(shí)驗(yàn)通過體外培養(yǎng)MSCs，了解其生長(zhǎng)生物學(xué)特性；探討其向肝系細(xì)胞分化的可能性，觀察肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor HGF)，堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast factor bFGF)及人胎肝細(xì)胞對(duì)培養(yǎng)的MSCs的誘導(dǎo)作用，尋找影響和調(diào)節(jié)MSCs向肝細(xì)胞分化的因素，探討體外誘導(dǎo)培養(yǎng)獲取肝細(xì)胞的可行性。以期為臨床肝細(xì)胞移植及生物人工肝治療提供新的細(xì)胞來源。 方法： 1．采用密度梯度離心法分離MSCs，反復(fù)貼壁法純化擴(kuò)增MSCs，鏡下觀察其形態(tài)學(xué)特點(diǎn)。 2．采用膠原酶Ⅰ型消化法分離人2-4月胚胎肝臟細(xì)胞，差速貼壁法純化肝臟細(xì)胞后培養(yǎng)。 3．通過加入一定濃度生長(zhǎng)因子HGF和bFGF對(duì)MSCs誘導(dǎo)培養(yǎng)，未加入生長(zhǎng)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。采用免疫細(xì)胞化學(xué)染色技術(shù)檢測(cè)誘導(dǎo)培養(yǎng)細(xì)胞的甲胎蛋白(alpha-fetoprotein AFP)、細(xì)胞角蛋白-18(cytokeratin-18 CK-18)、細(xì)胞角蛋白-19(cytokeratin-19 CK-19)等肝系細(xì)胞標(biāo)志物的表達(dá)情況及進(jìn)行糖原染色觀察誘導(dǎo)細(xì)胞的糖原合成及貯存功能，同時(shí)留取不同時(shí)段的細(xì)胞培養(yǎng)上清液用放射免疫法檢測(cè)AFP濃度以觀察誘導(dǎo)細(xì)胞的AFP合成及分泌功能。 4．MSCs標(biāo)記Hoechst33342后，將MSCs和人胎肝細(xì)胞直接混合培養(yǎng)和在雙室培養(yǎng)皿中半透膜隔開混合培養(yǎng)。胎肝細(xì)胞培養(yǎng)組及MSCs培養(yǎng)組作為對(duì)照組。觀察誘導(dǎo)細(xì)胞的形態(tài)學(xué)變化，采用免疫細(xì)胞化學(xué)染色技術(shù)檢測(cè)誘導(dǎo)培養(yǎng)
[Abstract]:Objective: bone marrow mesenchymal stem cells (MSCs) is a non-hematopoietic stem cell in bone marrow. Because of its self-renewal ability and multi-differentiation potential, it has shown a good application prospect in tissue and organ repair and regeneration. In this experiment, MSCs was cultured in vitro. To understand the biological characteristics of its growth; To investigate the possibility of hepatocyte growth factor differentiation. Induction of MSCs by basic fibroblast factor bFGF and human fetal hepatocytes. To find out the factors that influence and regulate the differentiation of MSCs into hepatocytes, and to explore the feasibility of inducing and culturing hepatocytes in vitro in order to provide a new cell source for clinical hepatocyte transplantation and bioartificial liver therapy. Methods: 1. MSCs were separated by density gradient centrifugation, then purified and amplified by repeated adherent method. The morphological characteristics of MSCs were observed under microscope. 2. The hepatocytes of 2-4 months embryo were isolated by collagenase 鈪，

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