本文關(guān)鍵詞：堿性成纖維細(xì)胞生長因子對胚胎干細(xì)胞來源暴式集落形成細(xì)胞形成的作用及其機(jī)制研究　出處：《濱州醫(yī)學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 集落形成細(xì)胞 堿性成纖維細(xì)胞生長因子 小鼠胚胎干細(xì)胞 Wnt Shh

【摘要】：目的:在擬胚體培養(yǎng)階段施加堿性成纖維細(xì)胞生長因子,驗(yàn)證堿性成纖維細(xì)胞生長因子對暴式集落形成細(xì)胞產(chǎn)生的調(diào)控作用及探討其作用機(jī)制。 方法:1.購買小鼠胚胎干細(xì)胞D3細(xì)胞系,取第3-5代小鼠原代胚胎成纖維細(xì)胞,加入新配制含絲裂霉素C的DMEM生長培養(yǎng)基孵育2.5 h,使飼養(yǎng)層細(xì)胞失去增殖能力;加入胰酶消化適度,離心后制成單細(xì)胞懸液,以10×10~4個/cm~2的密度種至用明膠包被的培養(yǎng)瓶中,放入孵箱內(nèi)培養(yǎng)24 h之后使用。復(fù)蘇胚胎干細(xì)胞D3細(xì)胞并將其接種于飼養(yǎng)層細(xì)胞之上。培養(yǎng)至一定時期后傳代并及時凍存。 2.在擬胚體培養(yǎng)階段按培養(yǎng)基成分不同分為4組:對照組(標(biāo)準(zhǔn)培養(yǎng)基+血管內(nèi)皮生長因子+干細(xì)胞因子組)、5μg/L bFGF組(標(biāo)準(zhǔn)培養(yǎng)基+血管內(nèi)皮生長因子+干細(xì)胞因子+5μg/L bFGF組)、10μg/L bFGF組(標(biāo)準(zhǔn)培養(yǎng)基+血管內(nèi)皮生長因子+干細(xì)胞因子+10μg/L bFGF組)、15μg/L bFGF組(標(biāo)準(zhǔn)培養(yǎng)基+血管內(nèi)皮生長因子+干細(xì)胞因子+15μg/L bFGF組)。各組于培養(yǎng)3、4、5、6d時分別計(jì)數(shù)克隆形成數(shù),通過免疫熒光法及IMAGE-PRO PLUS圖像分析系統(tǒng)檢測集落形成細(xì)胞Flk-1的表達(dá)情況,以此來篩選其最佳作用濃度。3.通過免疫熒光法檢測集落形成細(xì)胞相關(guān)表面標(biāo)志(Flk-1、CD133、CD34)的表達(dá)情況。4.通過免疫熒光法檢測相關(guān)通路的受體表達(dá)情況,通過RT-PCR法檢測相關(guān)信號通路特異性靶基因(Axin2、Gli)的表達(dá)情況,從而來進(jìn)一步探討bFGF發(fā)揮作用的相關(guān)機(jī)制。 結(jié)果:1.成功制備小鼠的胚胎成纖維細(xì)胞;2.擴(kuò)增并凍存大量的胚胎干細(xì)胞,并證明擴(kuò)增的胚胎干細(xì)胞具有全能性;3.培養(yǎng)體系中加入bFGF后,有大量克隆形成,克隆Flk-1呈陽性表達(dá),不同的濃度梯度克隆形成數(shù)量不同,且Flk-1的表達(dá)強(qiáng)度不同,其中10μg/L bFGF組克隆形成數(shù)量最多且Flk-1的表達(dá)最強(qiáng)(P 0.01);3.在BL-CFC生成過程中,Flk-1逐漸減弱,而CD133,CD34的表達(dá)逐漸增強(qiáng);4.在BL-CFC形成過程中,Wnt, Shh信號通路的表面受體表達(dá)逐漸增強(qiáng),其Wnt通路的特異性靶基因(Axin2)的表達(dá)也逐漸增強(qiáng)。 結(jié)論:1.成功制備小鼠的胚胎成纖維細(xì)胞,擴(kuò)增并凍存了大量的胚胎干細(xì)胞; 2. bFGF促進(jìn)了BL-CFC的形成,其最佳濃度為10μg/L; 3.形成的BL-CFC能夠向造血干/祖細(xì)胞方向分化; 4.在BL-CFC形成過程中,Wnt, Shh信號通路的表面受體表達(dá)逐漸增強(qiáng),Wnt通路的特異性靶基因(Axin2)的表達(dá)也逐漸增強(qiáng),初步證明bFGF促進(jìn)了BL-CFC的形成的作用機(jī)制與Wnt通路的激活相關(guān)。
[Abstract]:Objective : To investigate the effect of basic fibroblast growth factor ( bFGF ) on the formation of cells and explore its mechanism of action by applying basic fibroblast growth factor ( bFGF ) to the culture stage . Methods : 1 . Mouse embryonic stem cell D3 cell line was purchased , and the 3 - 5 mouse primary embryonic fibroblasts were cultured . The cells were cultured for 2.5 hours in DMEM medium supplemented with mitomycin C . 2 . The culture medium was divided into 4 groups according to the culture medium composition : control group ( standard medium + vascular endothelial growth factor + stem cell factor group ) , 5 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 5 渭g / L bFGF group ) , 10 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 10 渭g / L bFGF group ) , 15 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 15 渭g / L bFGF group ) . The expression of Flk - 1 , CD133 , CD34 was detected by immunofluorescence and IMAGE - PRO PLUS image analysis system . The expression of Flk - 1 , CD133 , CD34 was detected by immunofluorescence assay . Results : 1 . The mouse embryonic fibroblasts were successfully prepared ; 2 . Large amount of embryonic stem cells were amplified and frozen , and the expression intensity of Flk - 1 was higher than that of Flk - 1 . The expression intensity of Flk - 1 was different , and the expression intensity of Flk - 1 was significantly increased in the formation of BL - CFC . Conclusion : 1 . Mouse embryonic fibroblasts were successfully prepared , and a large number of embryonic stem cells were amplified and frozen . 2 . bFGF promoted BL - CFC formation , and the optimal concentration was 10 渭g / L . 3 . BL - CFC can differentiate into hematopoietic stem / progenitor cells . 4 . During the formation of BL - CFC , the expression of Wnt signaling pathway and Wnt signaling pathway gradually increased , and the expression of Wnt signaling pathway was gradually enhanced . The mechanism of bFGF promoting the formation of BL - CFC was associated with the activation of Wnt pathway .

【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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