本文選題：內(nèi)皮祖細(xì)胞 + 細(xì)胞培養(yǎng)��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2009年博士論文

【摘要】：第一部分人外周血內(nèi)皮祖細(xì)胞的分離培養(yǎng)與鑒定 目的:探討人外周血來源的內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)體外誘導(dǎo)培養(yǎng)和鑒定的方法,以期證實(shí)人外周血是基礎(chǔ)研究與臨床實(shí)踐的一個(gè)理想的內(nèi)皮祖細(xì)胞來源,并且建立一個(gè)內(nèi)皮祖細(xì)胞培養(yǎng)與鑒定有效的技術(shù)平臺(tái)。 方法:密度梯度離心法獲取健康成年男性外周血單個(gè)核細(xì)胞(peripheral bloodmononuclear cells,PBMNCs),接種在人纖維連接蛋白預(yù)先包被的培養(yǎng)板培養(yǎng),用EndoCult~(TM)內(nèi)皮祖細(xì)胞培養(yǎng)基誘導(dǎo)培養(yǎng),觀察內(nèi)皮祖細(xì)胞集落形成單位,收集貼壁細(xì)胞,根據(jù)內(nèi)皮祖細(xì)胞的特性,通過倒置熒光顯微鏡、流式細(xì)胞術(shù)、RT-PCR 3種方法鑒定內(nèi)皮祖細(xì)胞。 結(jié)果:從成人外周血分離獲得的PBMNCs經(jīng)體外誘導(dǎo)培養(yǎng),可以形成內(nèi)皮祖細(xì)胞的早期集落形成單位,表達(dá)內(nèi)皮祖細(xì)胞的相對(duì)特異性抗原,包括AC133、CD34和VEGFR-2,并顯示出具有能內(nèi)吞乙�；兔芏戎鞍�(acetylate low-densitylipoprotein,acLDL)和結(jié)合荊豆凝集素(ulex europaeus agglutinin-1 lectin,UEA-1lectin)的功能,同時(shí)可表達(dá)內(nèi)皮源型一氧化氮合酶基因(endothelial nitric oxidesynthase,eNOS),所誘導(dǎo)培養(yǎng)的細(xì)胞具有內(nèi)皮祖細(xì)胞的特征,鑒定為內(nèi)皮祖細(xì)胞。 結(jié)論:通過EndoCult~(TM)內(nèi)皮祖細(xì)胞培養(yǎng)基及其標(biāo)準(zhǔn)操作流程可以在人外周血中誘導(dǎo)培養(yǎng)EPCs,培養(yǎng)出的EPCs可表達(dá)內(nèi)皮祖細(xì)胞相對(duì)特異性抗原,包括AC133、CD34利VEGFR-2及具有內(nèi)皮細(xì)胞的內(nèi)吞乙�；兔芏戎鞍缀徒Y(jié)合荊豆凝集素,以及eNOS基因表達(dá)的功能。eNOS基因表達(dá)的檢測(cè)或可簡(jiǎn)化EPCs的鑒定。我們的實(shí)驗(yàn)為EPCs的分離鑒定方法提供了借鑒。 第二部分C反應(yīng)蛋白對(duì)內(nèi)皮祖細(xì)胞白介素8表達(dá)的影響及其機(jī)制 目的:血管內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)是血管內(nèi)皮細(xì)胞的前體細(xì)胞,不僅參與胚胎血管生成,也參與出生后的血管生發(fā)過程。C反應(yīng)蛋白(C-reactive protein,CRP)最初被認(rèn)為只是一種炎癥標(biāo)志物,并不直接參與炎癥反應(yīng)過程,但新近的證據(jù)證實(shí)在粥樣硬化性心血管疾病的發(fā)生和發(fā)展中,CRP可能參與了這一慢性炎癥損傷過程。白細(xì)胞介素8(Interleukin-8,IL-8)是由內(nèi)皮祖細(xì)胞分泌的一個(gè)重要的促血管新生因子。本研究旨在探討C反應(yīng)蛋白對(duì)內(nèi)皮祖細(xì)胞IL-8表達(dá)的影響并探討其可能的機(jī)制。 方法:以不同濃度(5～25μg/ml))人重組C反應(yīng)蛋白及不同時(shí)間段(3～48 hours)刺激人外周血分離培養(yǎng)的內(nèi)皮祖細(xì)胞,檢測(cè)其培養(yǎng)液上清IL-8因子的水平和細(xì)胞IL-8mRNA的水平�？笴D16、CD32抗體和P38-MAPK信號(hào)通路抑制劑預(yù)處理內(nèi)皮祖細(xì)胞后,再檢測(cè)CRP對(duì)其IL-8表達(dá)水平的影響。 結(jié)果:不同濃度的CRP加入到EPCs培養(yǎng)基中混合培養(yǎng)內(nèi)皮祖細(xì)胞12 hours后,real-time PCR和ELISA檢測(cè)IL-8的表達(dá)水平,結(jié)果顯示CRP能抑制EPCs IL-8的表達(dá),并且這一效應(yīng)與CRP濃度相關(guān),10μg/ml的CRP即可顯著抑制IL-8的表達(dá),25μg/ml濃度的CRP作用更強(qiáng);P38-MAPK信號(hào)通路抑制劑SB203580及抗CD32抗體預(yù)處理可以部分阻斷CRP對(duì)內(nèi)皮祖細(xì)胞抑制IL-8分泌的作用;而抗CD16抗體預(yù)處理內(nèi)皮祖細(xì)胞則無(wú)此作用。 結(jié)論:C反應(yīng)蛋白直接抑制內(nèi)皮祖細(xì)胞IL-8的表達(dá),CD32可能是內(nèi)皮祖細(xì)胞表面CRP的受體之一,P38-MAPK信號(hào)傳導(dǎo)通路可能是CRP作用于EPCs的信號(hào)轉(zhuǎn)導(dǎo)通路之一。
[Abstract]:Part 1 isolation, culture and identification of endothelial progenitor cells from human peripheral blood
Objective: To explore the methods of inducing culture and identification of endothelial progenitor cells (EPCs) from human peripheral blood in vitro, in order to prove that human peripheral blood is an ideal source of endothelial progenitor cells in basic and clinical practice, and to establish an effective technical platform for the culture and identification of inner skin progenitor cells.
Methods: density gradient centrifugation was used to obtain the peripheral blood mononuclear cells (peripheral bloodmononuclear cells, PBMNCs) of healthy adult male, inoculated with the culture plate of human fibronectin prepackaged by human fibronectin, and used EndoCult~ (TM) endothelial progenitor cell culture medium to induce culture, observe the colony forming unit of endothelial progenitor cells and collect the adherent cells. The characteristics of endothelial progenitor cells were identified by inverted fluorescence microscopy, flow cytometry and RT-PCR. 3 methods were used to identify endothelial progenitor cells.
Results: the PBMNCs obtained from the adult peripheral blood was induced and cultured in vitro to form an early colony forming unit of endothelial progenitor cells, expressing the relative specific antigens of endothelial progenitor cells, including AC133, CD34 and VEGFR-2, and showing the presence of acetylate Low-densitylipoprotein, acLDL, and binding of endocytic acetylated lipoprotein (acLDL). The function of Ulex europaeus agglutinin-1 lectin (UEA-1lectin) can also express the endothelial nitric oxide synthase gene (endothelial nitric oxidesynthase, eNOS). The induced cells have the characteristics of endothelial progenitor cells and are identified as inner skin progenitor cells.
Conclusion: EndoCult~ (TM) endothelial progenitor cell culture medium and its standard operation process can be induced to induce EPCs in human peripheral blood. The cultured EPCs can express the relative specific antigen of endothelial progenitor cells, including AC133, CD34 Li VEGFR-2, endothelium endocyacetylation low density lipoprotein and binding bean agglutinin with endothelial cells, and eNOS base. The detection of.ENOS gene expression can simplify the identification of EPCs because of the expression function. Our experiments provide a reference for the isolation and identification of EPCs.
The second part is the effect of C reactive protein on the expression of interleukin 8 in endothelial progenitor cells and its mechanism.
Objective: endothelial progenitor cells (EPCs) is a precursor cell of vascular endothelial cells, which not only participates in the angiogenesis of embryo, but also participates in the.C reactive protein (C-reactive protein, CRP) in the process of postnatal angiogenesis (C-reactive protein, CRP), which was originally considered as an inflammatory marker, not directly involved in the process of inflammation, but recently The evidence suggests that CRP may be involved in the process of chronic inflammatory damage in the occurrence and development of atherosclerotic cardiovascular disease. Interleukin 8 (Interleukin-8, IL-8) is an important neovascularization factor secreted by inner skin progenitor cells. The aim of this study is to explore the effect of C anti stress protein on the expression of IL-8 in endothelial progenitor cells. To discuss the possible mechanism.
Methods: the endothelial progenitor cells isolated from human peripheral blood were stimulated by human recombinant C reaction protein and different time periods (3~48 hours) in different concentrations (5~25 g/ml). The level of IL-8 factor and the level of cell IL-8mRNA in the culture liquid supernatant were detected. The anti CD16, CD32 antibody and P38-MAPK signal pathway inhibitors pretreated the endothelial progenitor cells and then detected CR The effect of P on the level of IL-8 expression.
Results: after adding different concentrations of CRP to EPCs medium, the expression level of IL-8 was detected by real-time PCR and ELISA. The results showed that CRP could inhibit the expression of EPCs IL-8, and this effect was related to CRP concentration. The expression of 10 micron g/ml could inhibit the expression of IL-8 significantly, and the effect of 25 micron concentration was stronger. The preconditioning of MAPK signaling pathway inhibitor SB203580 and anti CD32 antibody can partially block the inhibitory effect of CRP on the inhibition of IL-8 secretion by endothelial progenitor cells, while the anti CD16 antibody pretreated endothelial progenitor cells have no effect.
Conclusion: C reactive protein directly inhibits the expression of IL-8 in endothelial progenitor cells, and CD32 may be one of the CRP receptors on the surface of endothelial progenitor cells. The P38-MAPK signaling pathway may be one of the signal transduction pathways of CRP in EPCs.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王佐;童中藝;周曉峰;姜志勝;唐朝克;宋硯明;田永鳳;;微孔法分離大鼠骨髓內(nèi)皮祖細(xì)胞[J];生物化學(xué)與生物物理進(jìn)展;2007年07期

，

本文編號(hào)：1949513