本文關(guān)鍵詞： 胚胎干細(xì)胞 血管平滑肌細(xì)胞 血小板源性生長(zhǎng)因子　出處：《南昌大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:通過選擇適當(dāng)?shù)恼T導(dǎo)劑,探討胚胎干細(xì)胞體外向血管平滑肌細(xì)胞分化的趨勢(shì)。觀察胚胎干細(xì)胞在體外不同條件下定向誘導(dǎo)分化血管平滑肌細(xì)胞的能力。 方法:實(shí)驗(yàn)于2006年8月至2007年2月在南昌大學(xué)第二附屬醫(yī)院血液病研究所完成。①動(dòng)物及細(xì)胞系:清潔級(jí)孕12.5天的昆明小白鼠1只,實(shí)驗(yàn)過程中對(duì)動(dòng)物的處置符合動(dòng)物倫理學(xué)標(biāo)準(zhǔn);小鼠胚胎干細(xì)胞系129X/SvJ由美國(guó)ATCC提供。②實(shí)驗(yàn)方法:從12.5天的胎鼠中分離出原代胚胎成纖維細(xì)胞,當(dāng)原代胚胎成纖維細(xì)胞傳至3～5代時(shí),更換為含體積分?jǐn)?shù)為0.15胎牛血清的DMEM高糖培養(yǎng)基,24小時(shí)后收集培養(yǎng)液,加入條件培養(yǎng)基。用其對(duì)小鼠胚胎干細(xì)胞進(jìn)行體外培養(yǎng),傳代時(shí)用差速貼壁法分離去除已分化的胚胎干細(xì)胞,經(jīng)過懸滴-懸浮培養(yǎng),構(gòu)建擬胚體分化模型。設(shè)立3組,各組均置于0.1%明膠處理過的T25培養(yǎng)瓶中,每瓶加入50個(gè)擬胚體,均勻分布于培養(yǎng)瓶底,使擬胚體貼壁生長(zhǎng)。誘導(dǎo)組7～10天加入10-9mol/L全反式維甲酸和3μg/L轉(zhuǎn)化生長(zhǎng)因子β1,10～21天加入20μg/L血小板源性生長(zhǎng)因子,血清對(duì)照組僅加入去生長(zhǎng)因子胎牛血清,全反式維甲酸組加入全反式維甲酸。誘導(dǎo)21天的擬胚體,用胰蛋白酶和膠原酶Ⅱ聯(lián)合消化為單個(gè)細(xì)胞,再加入20μg/L血小板源性生長(zhǎng)因子繼續(xù)誘導(dǎo)7天。③實(shí)驗(yàn)評(píng)估:應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)誘導(dǎo)細(xì)胞血管平滑肌肌動(dòng)蛋白、血管平滑肌肌球蛋白重鏈基因的表達(dá),通過免疫細(xì)胞化學(xué)技術(shù)檢測(cè)血管平滑肌肌動(dòng)蛋白的表達(dá)來鑒定細(xì)胞性質(zhì)。 結(jié)果:①胚胎干細(xì)胞生長(zhǎng)及擬胚體誘導(dǎo)分化:胚胎干細(xì)胞在體外能自發(fā)形成擬胚體,經(jīng)過不同生長(zhǎng)因子的分階段聯(lián)合誘導(dǎo),擬胚體周圍出現(xiàn)大量的梭形細(xì)胞。②RT-PCR檢測(cè):擬胚體血管平滑肌肌動(dòng)蛋白和血管平滑肌肌球蛋白重鏈基因均呈陽(yáng)性表達(dá)。③免疫細(xì)胞化學(xué)檢測(cè):熒光顯微鏡下,羅丹明染色細(xì)胞漿呈紅色熒光,并可見肌絲結(jié)構(gòu);DAPI染色細(xì)胞核呈藍(lán)色熒光。誘導(dǎo)組血管平滑肌肌動(dòng)蛋白陽(yáng)性率明顯高于血清對(duì)照組和全反式維甲酸組(P0.01)。 結(jié)論:①在條件培養(yǎng)基無飼養(yǎng)層上可大量擴(kuò)增胚胎干細(xì)胞并保持其未分化狀態(tài)。②胚胎干細(xì)胞經(jīng)全反式維甲酸、轉(zhuǎn)化生長(zhǎng)因子β1以及血小板源性生長(zhǎng)因子分階段聯(lián)合誘導(dǎo)后,可分化為血管平滑肌細(xì)胞且純度較高。
[Abstract]:Aim: to investigate the tendency of embryonic stem cells to differentiate into vascular smooth muscle cells (VSMCs) in vitro by selecting suitable inducers, and to observe the ability of embryonic stem cells to induce differentiation of vascular smooth muscle cells (VSMCs) under different conditions in vitro. Methods: from August 2006 to February 2007, 1 animal and cell line were completed in the Institute of Hematology, second affiliated Hospital of Nanchang University. The treatment of animals in the course of the experiment was in accordance with the standard of animal ethics; the mouse embryonic stem cell line 129X / SvJ was provided by the ATCC of the United States with the method of 2.2. Primary embryonic fibroblasts were isolated from 12.5-day-old fetal mice. When the primary embryonic fibroblasts were transferred to 3 ~ 5 passages, the DMEM medium containing 0.15 fetal bovine serum was replaced with high sugar medium for 24 hours, then the medium was collected and added to the conditioned medium. The mouse embryonic stem cells were cultured in vitro. The differentiated embryonic stem cells were separated and removed by differential adherent method during passage. The model of embryoid differentiation was established by suspension and suspension culture. Three groups were set up and each group was placed in the culture flask of T25 treated with 0.1% gelatin, 50 embryoid bodies were added to each bottle. In the induction group, 10-9 mol / L all-trans retinoic acid and 3 渭 g / L transforming growth factor 尾 _ 1 / L were added to the platelet-derived growth factor for 10 ~ 21 days, while the serum control group was added only to the bovine serum derived from growth factor. The all-trans retinoic acid group was added with all-trans retinoic acid. The embryoid body was induced for 21 days and digested into a single cell by trypsin and collagenase 鈪，

本文編號(hào)：1527399