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

【摘要】： 目的:系統(tǒng)的研究小鼠胚胎干細(xì)胞的提取、體外培養(yǎng)擴(kuò)增和向成骨細(xì)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(xì)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(jìn)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(xì)胞團(tuán)集落進(jìn)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(jìn)行分化抑制培養(yǎng),并進(jìn)行傳代和體外擴(kuò)增。采用堿性磷酸酶染色、核型分析、類胚體實(shí)驗(yàn)對擴(kuò)增細(xì)胞進(jìn)行ESC鑒定。將小鼠5d齡ES-D3細(xì)胞源的類胚體(EBs)單細(xì)胞,按5×104/mL接種12孔細(xì)胞培養(yǎng)板,在DMEM基礎(chǔ)培養(yǎng)基中使EBs單細(xì)胞貼壁培養(yǎng)。于第14～21天時(shí),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(shí)用1%茜素紅染色顯示陽性細(xì)胞,計(jì)算成骨細(xì)胞誘導(dǎo)形成率,數(shù)據(jù)用方差分析和多重比較檢驗(yàn)。 結(jié)果:成纖維細(xì)胞接種半小時(shí)后貼壁,可見細(xì)小突起長出,細(xì)胞呈梭形,核圓形。24小時(shí)后即可連生。ES細(xì)胞推開飼養(yǎng)層細(xì)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(xì)胞。細(xì)胞呈團(tuán)狀緊密排列,內(nèi)部單個(gè)細(xì)胞辨別不清,每個(gè)細(xì)胞呈圓形,部分呈梭形,體積小,核大,胞漿少。細(xì)胞堿性磷酸酶染色呈強(qiáng)陽性,為二倍體核型,可形成類胚體。表明培養(yǎng)擴(kuò)增后的細(xì)胞具有ESC的典型特征。在EBs單細(xì)胞貼壁培養(yǎng)的第14～21d加入成骨誘導(dǎo)劑后,細(xì)胞形態(tài)發(fā)生改變,原來的球形細(xì)胞逐漸發(fā)出突起,變?yōu)槎嘟切位蚨趟笮蔚念惓衫w維細(xì)胞樣,茜素紅染色為紅色,證明已轉(zhuǎn)化為成骨細(xì)胞。 結(jié)論:1.原代成纖維細(xì)胞分離方法簡單,來源方便,細(xì)胞貼壁快,增殖迅速,作為飼養(yǎng)層能促進(jìn)ES細(xì)胞的增殖并抑制其分化,因而非常適用于ES細(xì)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(xì)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 沈干,汪錚,叢笑倩,曹誼林;組織工程中的新型種子細(xì)胞——胚胎干細(xì)胞[J];國外醫(yī)學(xué).生物醫(yī)學(xué)工程分冊;2001年03期

2 唐康來,李起鴻,楊柳;骨、軟骨組織工程種子細(xì)胞及其免疫學(xué)相關(guān)研究進(jìn)展[J];免疫學(xué)雜志;2002年S1期

3 郭雨霽,高英茂,韓愛民;胚胎干細(xì)胞的分離與培養(yǎng)[J];生命的化學(xué);2002年06期

4 楊柳,段小軍;骨組織工程學(xué)中種子細(xì)胞研究的前沿問題[J];中國臨床康復(fù);2002年04期

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