本文關(guān)鍵詞：Galectin-3調(diào)控Tip血管內(nèi)皮細(xì)胞塑管效應(yīng)的機(jī)制研究　出處：《昆明醫(yī)科大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 內(nèi)皮祖細(xì)胞 內(nèi)皮細(xì)胞 Tip血管內(nèi)皮細(xì)胞 Galectin-3 Tip血管內(nèi)皮細(xì)胞 增殖

【摘要】：第一部分人外周血內(nèi)皮祖細(xì)胞及Tip血管內(nèi)皮細(xì)胞的分離培養(yǎng)與鑒定 目的：探討人外周血內(nèi)皮祖細(xì)胞的分離與培養(yǎng)方法,研究人外周血內(nèi)皮祖細(xì)胞的體外誘導(dǎo)培養(yǎng)條件及其生物學(xué)活性,擬證實(shí)人外周血是內(nèi)皮祖細(xì)胞除了骨髓以外的另一個(gè)理想來(lái)源。在一定的體外培養(yǎng)條件下,外周血內(nèi)皮祖細(xì)胞還可定向誘導(dǎo)分化為T(mén)ip血管內(nèi)皮細(xì)胞,不僅為下一步實(shí)驗(yàn)研究提供所需的細(xì)胞來(lái)源,還為干細(xì)胞源性血管新生機(jī)制提供可靠的實(shí)驗(yàn)室依據(jù)。 方法：采用Ficoll密度梯度離心法分離人外周血單個(gè)核細(xì)胞,將其接種在預(yù)先包被有人纖維連接蛋白的培養(yǎng)瓶中培養(yǎng)。每日于倒置相差顯微鏡下觀察細(xì)胞的生長(zhǎng)形態(tài)及內(nèi)皮祖細(xì)胞集落形成形態(tài),每隔3-4天換一次液,培養(yǎng)7天后收集貼壁細(xì)胞,根據(jù)內(nèi)皮祖細(xì)胞的特性,用流式細(xì)胞儀檢測(cè)內(nèi)皮祖細(xì)胞特異性抗原CD34、CD133及VEGFR等表面標(biāo)志物,以鑒定培養(yǎng)后的細(xì)胞主要為內(nèi)皮祖細(xì)胞。培養(yǎng)至21天時(shí)倒置相差顯微鏡下觀察和鑒定Tip血管內(nèi)皮細(xì)胞形態(tài),并收集該貼壁細(xì)胞進(jìn)行RT-PCR實(shí)驗(yàn)檢測(cè)Tip血管內(nèi)皮細(xì)胞中vWF、eNOS等血管內(nèi)皮細(xì)胞系特異性基因的表達(dá)情況。 結(jié)果：分離獲得的單個(gè)核細(xì)胞培養(yǎng)4天后多數(shù)呈貼壁生長(zhǎng),7天后形成梭狀的內(nèi)皮樣細(xì)胞,部分細(xì)胞積聚成團(tuán)形成克隆集落,流式細(xì)胞儀鑒定該細(xì)胞表達(dá)的內(nèi)皮祖細(xì)胞特異性抗原CD34、CD133及VEGFR分別為(68.9±2.34)%、(81.7±1.50)%、(97.1±2.01)%。繼續(xù)培養(yǎng)21天后的細(xì)胞呈鵝卵石樣改變,并伸出偽足相互連接形成類(lèi)似血管腔樣結(jié)構(gòu),形態(tài)學(xué)表現(xiàn)為T(mén)ip血管內(nèi)皮細(xì)胞的形態(tài)特點(diǎn),RT-PCR實(shí)驗(yàn)檢測(cè)該細(xì)胞有血管內(nèi)皮細(xì)胞系特異性基因vWF、eNOS的表達(dá)。 結(jié)論：可以成功從人外周血中分離具有增殖分化潛能的內(nèi)皮祖細(xì)胞,該細(xì)胞可形成內(nèi)皮祖細(xì)胞特有的集落形成單位,并表達(dá)內(nèi)皮祖細(xì)胞的相對(duì)特異性抗原CD34、CD133及VEGFR。同時(shí)經(jīng)過(guò)特定的體外誘導(dǎo)培養(yǎng)體系可將其擴(kuò)增并定向誘導(dǎo)分化為T(mén)ip血管內(nèi)皮細(xì)胞。 第二部分Galectin-3對(duì)外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞增殖能力的影響 目的：內(nèi)皮祖細(xì)胞是血管內(nèi)皮細(xì)胞的前體細(xì)胞,是一群具有游走特性,在生理或病理因素的刺激下,可從骨髓動(dòng)員到外周血,進(jìn)一步增殖分化并參與受損血管的修復(fù)的幼稚內(nèi)皮細(xì)胞。半乳糖凝集素-3(Galectin-3)是一種半乳糖結(jié)合蛋白,該蛋白的糖結(jié)構(gòu)識(shí)別域在血管生成的糖依賴(lài)過(guò)程中起著關(guān)鍵的作用。本實(shí)驗(yàn)觀察Galectin-3對(duì)外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞增殖能力的影響,為臨床干細(xì)胞治療慢性下肢動(dòng)脈缺血性疾病提供新的實(shí)驗(yàn)室依據(jù)。 方法：收集第一部分實(shí)驗(yàn)中培養(yǎng)21天后的貼壁Tip血管內(nèi)皮細(xì)胞,隨機(jī)將Galectin-3分為終濃度各濃度組(共6組)：在外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞的培養(yǎng)液中分別加入終濃度為0ug/ml,0.1ug/ml,1.0ug/ml,2.5ug/ml,5.0ug/m1和10ug/ml的Galectin-3培養(yǎng)24小時(shí)。MTT比色法觀察不同濃度Galectin-3對(duì)外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞增殖能力的影響。 結(jié)果：六組不同終濃度的Galectin-3對(duì)外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞增殖能力的影響有差別(F值=37.329,P0.01)。加入Galectin-3的各濃度組外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞的增殖能力均高于Oug/ml組,其中0.10ug/ml組,1.00ug/ml組及2.50ug/ml組與0ug/ml組比較無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),但5.0ug/ml組及10.0ug/ml組明顯高于0ug/ml組,0.10ug/ml組,1.00ug/ml組及2.50ug/ml組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);10.0ug/ml濃度組也明顯高于5.0ug/ml濃度組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞增殖能力與Galectin-3濃度間存在正相關(guān)關(guān)系(r=0.966),兩變量有高度相關(guān)關(guān)系。 結(jié)論：Galectin-3可促進(jìn)體外培養(yǎng)外周血內(nèi)皮祖細(xì)胞源性Tip血管內(nèi)皮細(xì)胞的增殖能力,是Tip血管內(nèi)皮細(xì)胞塑管效應(yīng)過(guò)程中重要的因子之一。
[Abstract]:Isolation , culture and identification of endothelial progenitor cells and vascular endothelial cells from human peripheral blood in the first part Objective : To study the isolation and culture of endothelial progenitor cells from human peripheral blood . It is suggested that human peripheral blood endothelial progenitor cells can induce differentiation into the endothelial progenitor cells in vitro . Methods : Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation . The growth morphology of endothelial progenitor cells and the formation of endothelial progenitor cells were observed under reversed phase contrast microscope . The surface markers of endothelial progenitor cells specific antigen CD34 , CD133 and VEGFR were detected by flow cytometry after 7 days . Results : After 4 days after the single core cell culture was isolated , most of the cells were adherent . After 7 days , spindle - shaped endothelium - like cells were formed . The specific antigens CD34 , CD133 and VEGFR were ( 68.9 鹵 2.34 ) % , ( 81.7 鹵 1.50 ) % and ( 97.1 鹵 0.01 ) % respectively . Conclusion : Endothelial progenitor cells with proliferative differentiation potential can be isolated from human peripheral blood successfully , which can form a specific colony forming unit of endothelial progenitor cells and express the relative specific antigens CD34 , CD133 and VEGFR of endothelial progenitor cells . Effect of the second part galectin - 3 on the proliferation of endothelial progenitor cells in peripheral vascular endothelial cells Objective : To investigate the effect of Galectin - 3 on the proliferation of vascular endothelial cells derived from vascular endothelial progenitor cells and to provide a new experimental basis for the treatment of chronic lower extremity arterial ischemic diseases by Galectin - 3 . Galectin - 3 is a kind of galactose binding protein . Methods : Galectin - 3 was randomly divided into final concentration groups ( group 6 ) . Galectin - 3 was cultured for 24 hours in the culture solution of peripheral vascular endothelial progenitor cells . The effects of Galectin - 3 on the proliferation of endothelial progenitor cells in peripheral blood were observed by MTT colorimetric method . Results : The effects of Galectin - 3 at different final concentrations on proliferation of endothelial progenitor cells in peripheral blood were different ( F = 37.329 , P0.01 ) . The proliferative capacity of endothelial progenitor cells in peripheral vascular endothelial cells was higher than that in Oug / ml group ( P0.05 ) . Conclusion : Galectin - 3 can promote the proliferation of endothelial progenitor cells derived from endothelial progenitor cells in vitro , which is one of the most important factors in the plastic tube effect of endothelial cells .

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 楊鏞;陸平;何曉明;楊國(guó)凱;羅開(kāi)元;段志泉;時(shí)德;;人自體干細(xì)胞移植在重癥肢體缺血血流重建中的療效與評(píng)價(jià)[J];中國(guó)普外基礎(chǔ)與臨床雜志;2009年02期

2 楊國(guó)凱;楊鏞;何曉明;羅開(kāi)元;萬(wàn)嘉;楊光;李國(guó)劍;馬震寰;陸平;;外周血干細(xì)胞移植治療血栓閉塞性脈管炎[J];中國(guó)微創(chuàng)外科雜志;2009年09期

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本文編號(hào)：1412548