發(fā)布時間：2018-02-23 19:47

  本文關(guān)鍵詞： 胚胎干細胞 血管平滑肌細胞 血小板源性生長因子　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?shù)恼T導(dǎo)劑,探討胚胎干細胞體外向血管平滑肌細胞分化的趨勢。觀察胚胎干細胞在體外不同條件下定向誘導(dǎo)分化血管平滑肌細胞的能力。 方法:實驗于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動物及細胞系:清潔級孕12.5天的昆明小白鼠1只,實驗過程中對動物的處置符合動物倫理學(xué)標準;小鼠胚胎干細胞系129X/SvJ由美國ATCC提供。②實驗方法:從12.5天的胎鼠中分離出原代胚胎成纖維細胞,當原代胚胎成纖維細胞傳至3～5代時,更換為含體積分數(shù)為0.15胎牛血清的DMEM高糖培養(yǎng)基,24小時后收集培養(yǎng)液,加入條件培養(yǎng)基。用其對小鼠胚胎干細胞進行體外培養(yǎng),傳代時用差速貼壁法分離去除已分化的胚胎干細胞,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個擬胚體,均勻分布于培養(yǎng)瓶底,使擬胚體貼壁生長。誘導(dǎo)組7～10天加入10-9mol/L全反式維甲酸和3μg/L轉(zhuǎn)化生長因子β1,10～21天加入20μg/L血小板源性生長因子,血清對照組僅加入去生長因子胎牛血清,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)合消化為單個細胞,再加入20μg/L血小板源性生長因子繼續(xù)誘導(dǎo)7天。③實驗評估:應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測誘導(dǎo)細胞血管平滑肌肌動蛋白、血管平滑肌肌球蛋白重鏈基因的表達,通過免疫細胞化學(xué)技術(shù)檢測血管平滑肌肌動蛋白的表達來鑒定細胞性質(zhì)。 結(jié)果:①胚胎干細胞生長及擬胚體誘導(dǎo)分化:胚胎干細胞在體外能自發(fā)形成擬胚體,經(jīng)過不同生長因子的分階段聯(lián)合誘導(dǎo),擬胚體周圍出現(xiàn)大量的梭形細胞。②RT-PCR檢測:擬胚體血管平滑肌肌動蛋白和血管平滑肌肌球蛋白重鏈基因均呈陽性表達。③免疫細胞化學(xué)檢測:熒光顯微鏡下,羅丹明染色細胞漿呈紅色熒光,并可見肌絲結(jié)構(gòu);DAPI染色細胞核呈藍色熒光。誘導(dǎo)組血管平滑肌肌動蛋白陽性率明顯高于血清對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)層上可大量擴增胚胎干細胞并保持其未分化狀態(tài)。②胚胎干細胞經(jīng)全反式維甲酸、轉(zhuǎn)化生長因子β1以及血小板源性生長因子分階段聯(lián)合誘導(dǎo)后,可分化為血管平滑肌細胞且純度較高。
[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：1527399