本文選題：胚胎干細胞　切入點：誘導(dǎo)　出處：《山西醫(yī)科大學》2006年碩士論文　論文類型：學位論文

【摘要】： 目的:系統(tǒng)的研究小鼠胚胎干細胞的提取、體外培養(yǎng)擴增和向成骨細胞定向誘導(dǎo)分化。 方法:用13.5d左右的昆明小鼠胚胎,采用常溫消化法制備小鼠胚胎原代成纖維細胞(PMEF),加入專用的DMEM培養(yǎng)液,經(jīng)絲裂霉素C處理后成為飼養(yǎng)層。在昆明小鼠受精后第4天,從子宮中取囊胚進行體外培養(yǎng),4～5天后,獲得大的巢式生長集落,使用0.25%胰蛋白酶-0.02% EDTA,采用兩步離散法,對內(nèi)細胞團集落進行離散,接種在新鮮制備的飼養(yǎng)層體系中,加入適宜的含有白血病抑制因子(LlF)的培養(yǎng)基進行分化抑制培養(yǎng),并進行傳代和體外擴增。采用堿性磷酸酶染色、核型分析、類胚體實驗對擴增細胞進行ESC鑒定。將小鼠5d齡ES-D3細胞源的類胚體(EBs)單細胞,按5×104/mL接種12孔細胞培養(yǎng)板,在DMEM基礎(chǔ)培養(yǎng)基中使EBs單細胞貼壁培養(yǎng)。于第14～21天時,分別添加50μg/mL維生素C、50 mmol/Lβ-磷酸甘油、1μmol/L地塞米松(A組), 50μg/mL維生素C、50 mmol/Lβ-磷酸甘油、1μmol/L地塞米松、10ng/mL骨形成蛋白2(B組),并設(shè)不添加誘導(dǎo)劑對照組(C組)。第22天時用1%茜素紅染色顯示陽性細胞,計算成骨細胞誘導(dǎo)形成率,數(shù)據(jù)用方差分析和多重比較檢驗。 結(jié)果:成纖維細胞接種半小時后貼壁,可見細小突起長出,細胞呈梭形,核圓形。24小時后即可連生。ES細胞推開飼養(yǎng)層細胞生長,形成集落,集落邊緣光滑清晰,有的集落邊緣可見小的散在的ES細胞。細胞呈團狀緊密排列,內(nèi)部單個細胞辨別不清,每個細胞呈圓形,部分呈梭形,體積小,核大,胞漿少。細胞堿性磷酸酶染色呈強陽性,為二倍體核型,可形成類胚體。表明培養(yǎng)擴增后的細胞具有ESC的典型特征。在EBs單細胞貼壁培養(yǎng)的第14～21d加入成骨誘導(dǎo)劑后,細胞形態(tài)發(fā)生改變,原來的球形細胞逐漸發(fā)出突起,變?yōu)槎嘟切位蚨趟笮蔚念惓衫w維細胞樣,茜素紅染色為紅色,證明已轉(zhuǎn)化為成骨細胞。 結(jié)論:1.原代成纖維細胞分離方法簡單,來源方便,細胞貼壁快,增殖迅速,作為飼養(yǎng)層能促進ES細胞的增殖并抑制其分化,因而非常適用于ES細胞的分離培養(yǎng)。2.維生素C、β-磷酸甘油、地塞米松、BMP-2組成的成骨誘導(dǎo)劑能夠誘導(dǎo)ESC分化為成骨細胞,誘導(dǎo)分化率為29%左右。
[Abstract]:Aim: to study systematically the extraction of mouse embryonic stem cells (ESCs), culture and amplification in vitro and differentiation into osteoblasts. Methods: Kunming mouse embryos of about 13.5 days were prepared by normal temperature digestion method. The primary fibroblasts of mouse embryos were prepared by normal temperature digestion method. The primary fibroblasts of mouse embryos were cultured in a special DMEM culture medium and then treated with mitomycin C to form a feeder layer after fertilization in Kunming mice on the 4th day after fertilization. After the blastocysts were cultured in vitro for 5 days, a large nest growth colony was obtained. 0.25% trypsin -0.02% EDTA was used to disperse the inner cell colony, and the colony was inoculated in the fresh feeder layer system. Suitable culture medium containing leukemia inhibitor LlF was added for differentiation inhibition culture, passage and in vitro amplification. Alkaline phosphatase staining and karyotype analysis were used. ESC identification of expanded cells was carried out by embryoid body experiment. Single cells of ES-D3 cells derived from ES-D3 cells of 5 days old in mice were inoculated with 12 well cell culture plate according to 5 脳 10 4 / mL, and EBs single cells were cultured in DMEM basic medium on day 14 to 21, respectively. 50 渭 g / mL vitamin C + 50 mmol/L 尾 -phosphoglycerol 1 渭 mol/L dexamethasone A group and 50 渭 g / mL vitamin C cor 50 mmol/L 尾 -phosphate glycerol phosphate 1 渭 mol/L dexamethasone 1 渭 mol/L dexamethasone 10 渭 mol/L / mL bone morphogenetic protein 2ng / mL group B were added respectively, and the control group C was stained with 1% alizarin red at 22 days after adding 50 渭 g / mL vitamin C and 10 渭 mol/L dexamethasone / mL bone morphogenetic protein 2ng / mL. Color positive cells, The osteoblast induction rate was calculated by ANOVA and multiple comparison test. Results: the fibroblasts adhered to the wall half an hour after inoculation, and the cells were spindle-shaped. After 24 hours of nuclear circle, they could push the feeder layer cells to grow, forming a colony, and the margin of the colony was smooth and clear. Small scattered es cells were seen on the edge of the colony. The cells were clustered and closely arranged, and the single cells were indistinguishable. Each cell was round, partly fusiform, small in size, large in nucleus, small in cytoplasm, and strongly positive for alkaline phosphatase staining. It is a diploid karyotype, which can form embryoid body. It shows that the expanded cells have the typical characteristics of ESC. After adding osteogenic inducer at 1421 days after EBs single cell adherent culture, the morphology of the cells changes and the original spherical cells gradually emit processes. It became polygonal or fusiform fibroblast-like, and alizarin red staining was red, which proved that it had been transformed into osteoblast. Conclusion the primary fibroblast isolation method is simple and convenient, the cells adhere to the wall quickly and proliferate rapidly. As the feeder layer, it can promote the proliferation and inhibit the differentiation of es cells. The osteogenic inducer composed of vitamin C, 尾 -glycerol phosphate and dexamethasone BMP-2 can induce the differentiation of ESC into osteoblasts and the differentiation rate is about 29%.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R329.2

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