發(fā)布時(shí)間：2018-11-21 19:29

【摘要】：背景:大鼠骨髓胚胎樣干細(xì)胞能表達(dá)胚胎干細(xì)胞標(biāo)志物,能夠分化形成三胚層來源細(xì)胞,并且植入機(jī)體后能改善心功能、組織心室重構(gòu),但是對(duì)于骨髓胚胎樣干細(xì)胞的生物學(xué)特性及分化潛能尚不完全知曉。目的:分析大鼠骨髓胚胎樣干細(xì)胞生物特性和分化潛能。方法:20只SD大鼠,采用階段特異性胚胎抗原1(SSAE-1)標(biāo)記骨髓單核細(xì)胞,利用流式細(xì)胞儀分選骨髓胚胎樣干細(xì)胞。采用電鏡掃描觀察骨髓胚胎樣干細(xì)胞形態(tài)、結(jié)構(gòu);免疫熒光化學(xué)染色測(cè)定胚胎干細(xì)胞轉(zhuǎn)錄因子(Oct4、Nanog、Sox2)在骨髓胚胎樣干細(xì)胞中的表達(dá),將骨髓胚胎樣干細(xì)胞放入纖維細(xì)胞飼養(yǎng)層進(jìn)行擴(kuò)增,誘導(dǎo)心肌和內(nèi)皮細(xì)胞,測(cè)定其特異蛋白表達(dá)。結(jié)果與結(jié)論:(1)采用SSEA-1標(biāo)記大鼠骨髓胚胎樣干細(xì)胞,對(duì)直徑為2-6μm細(xì)胞設(shè)門,大鼠骨髓胚胎樣干細(xì)胞呈現(xiàn)紡錘形,多邊形以及圓形多細(xì)胞形態(tài);體積較小;(2)透射電鏡下可見細(xì)胞核增大,細(xì)胞質(zhì)較少,含有少量的細(xì)胞器。(3)第2代大鼠骨髓胚胎樣干細(xì)胞免疫細(xì)胞化學(xué)染色下能測(cè)定到多潛能抗原標(biāo)志;隨傳代次數(shù)增加,細(xì)胞增殖能力出現(xiàn)下降趨勢(shì);(4)大鼠骨髓胚胎樣干細(xì)胞分化1周后心肌細(xì)胞平行排列,誘導(dǎo)2周后細(xì)胞形態(tài)發(fā)生明顯的變化,細(xì)胞相互連接。免疫熒光化學(xué)染色可見心肌特異蛋白cTnT以及Cx43表達(dá);大鼠骨髓胚胎干細(xì)胞分化1周后內(nèi)皮細(xì)胞呈現(xiàn)多角形、梭形。誘導(dǎo)2周后細(xì)胞相互連接,免疫細(xì)胞化學(xué)染色可見內(nèi)皮細(xì)胞特異蛋白VWF以及CD31表達(dá)。(5)結(jié)果提示:大鼠骨髓胚胎樣干細(xì)胞具備與骨髓間充質(zhì)干細(xì)胞類似的生物學(xué)特性,能表達(dá)干細(xì)胞轉(zhuǎn)錄因子,能夠在一定的條件下分化為心肌細(xì)胞和內(nèi)皮細(xì)胞,具有較高的臨床應(yīng)用價(jià)值。
[Abstract]:Background: rat bone marrow embryonic stem cells (BMSCs) can express embryonic stem cell markers, differentiate into tridermal derived cells, and improve cardiac function and ventricular remodeling after implantation. However, the biological characteristics and differentiation potential of bone marrow embryonic stem cells are not fully known. Objective: to analyze the biological characteristics and differentiation potential of rat bone marrow embryonic stem cells (BMSCs). Methods: bone marrow monocytes were labeled with stage specific embryonic antigen 1 (SSAE-1) in 20 SD rats and bone marrow embryonic stem cells were sorted by flow cytometry. The morphology and structure of bone marrow embryonic stem cells were observed by scanning electron microscope. The expression of embryonic stem cell transcription factor (Oct4,Nanog,Sox2) in bone marrow embryonic like stem cells was determined by immunofluorescence staining. Bone marrow embryonic stem cells were added to fibroblast feeder layer to induce myocardial and endothelial cells. Its specific protein expression was determined. Results and conclusion: (1) SSEA-1 was used to label embryonic stem cells of rat bone marrow, and the diameter of 2-6 渭 m cells was set up. The embryonic stem cells of rat bone marrow showed fusiform, polygonal and round multicellular morphology, and the size was small. (2) under the transmission electron microscope, the nucleus was enlarged, the cytoplasm was less and there were a few organelles. (3) the multipotential antigen markers could be detected by immunocytochemical staining of the second generation of rat bone marrow embryonic stem cells. With the increase of passage times, the proliferation of rat bone marrow embryonic stem cells decreased. (4) after 1 week of differentiation, the cardiomyocytes were arranged in parallel, and after 2 weeks of induction, the morphology of the cells changed obviously and the cells connected with each other. The expression of myocardial specific protein cTnT and Cx43 was observed by immunofluorescence staining, and the endothelial cells of rat bone marrow embryonic stem cells showed polygonal and fusiform shape after 1 week of differentiation. After 2 weeks of induction, the cells were connected to each other, and the expression of endothelial cell specific protein VWF and CD31 was observed by immunocytochemistry. (5) the results suggested that the embryonic stem cells of rat bone marrow had similar biological characteristics to those of mesenchymal stem cells. It can express stem cell transcription factors and can differentiate into cardiomyocytes and endothelial cells under certain conditions.
【作者單位】： 白城醫(yī)學(xué)高等�？茖W(xué)校;吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院;
【分類號(hào)】：R54

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