本文選題：內(nèi)皮祖細(xì)胞 + 細(xì)胞培養(yǎng)��； 參考：《復(fù)旦大學(xué)》2009年博士論文

【摘要】： 第一部分內(nèi)皮祖細(xì)胞的分離培養(yǎng)和鑒定 目的:利用實(shí)驗(yàn)室已建立的外周血內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)分離培養(yǎng)方案,建立EPCs的培養(yǎng)體系并進(jìn)行細(xì)胞表型和功能的檢測(cè)與鑒定,為后續(xù)的實(shí)驗(yàn)研究提供細(xì)胞來(lái)源。 方法:采集新西蘭大白兔靜脈外周血20ml,以密度梯度離心法獲得單個(gè)核細(xì)胞,將細(xì)胞放入涂有0.1%纖連蛋白的培養(yǎng)皿中,在加有20%胎牛血清(FBS)和EGM-2 SingleQuots的內(nèi)皮細(xì)胞基礎(chǔ)培養(yǎng)基-2(EBM-2)中培養(yǎng)。于培養(yǎng)第7d應(yīng)用免疫熒光檢測(cè)細(xì)胞標(biāo)志CD133,CD34,vWF,CD45抗原的表達(dá);利用DiI-acLDL和FITC-UEA-1吸收試驗(yàn)鑒定內(nèi)皮功能。 結(jié)果:單個(gè)核細(xì)胞在內(nèi)皮條件下培養(yǎng),48h后逐漸貼壁,第3d細(xì)胞呈梭形或不規(guī)則形,并逐漸變大,第7d梭形細(xì)胞增多,部分視野觀察到梭形細(xì)胞首尾相連形成條索樣結(jié)構(gòu)。熒光顯微鏡觀察CD133,CD34,vWF陽(yáng)性率分別為83.11±3.46%,82.87±4.66%,77.45±6.71%,CD45陰性。并且,有＞90%的細(xì)胞吸收DiI-ac LDL和FITC-UEA-1。 結(jié)論:應(yīng)用本研究方法從外周血途徑分離、培養(yǎng)和擴(kuò)增獲得EPC,具有內(nèi)皮祖細(xì)胞的特征和功能。 第二部分基質(zhì)細(xì)胞衍生因子-1對(duì)內(nèi)皮祖細(xì)胞的體外作用 目的:制備本實(shí)驗(yàn)室已凍存的病毒基因載體轉(zhuǎn)導(dǎo)細(xì)胞NIH3T3/SDF-1和NIH3T3/LacZ細(xì)胞并檢測(cè)細(xì)胞功能,利用其分泌基質(zhì)細(xì)胞衍生因子-1(SDF-1)的特點(diǎn)研究對(duì)體外培養(yǎng)、擴(kuò)增的EPC的影響。 方法:解凍NIH3T3細(xì)胞通過(guò)逆轉(zhuǎn)錄病毒轉(zhuǎn)導(dǎo)SDF-1α基因和LacZ基因后制作能表達(dá)SDF-1的NIH3T3細(xì)胞(NIH3T3/SDF-1)和表達(dá)LacZ的NIH3T3/LacZ細(xì)胞。用ELASA法測(cè)定NIH3T3細(xì)胞,NIH3T3/SDF-1細(xì)胞在體外SDF-1表達(dá)情況,及存在EPO(10IU/ml)是對(duì)細(xì)胞表達(dá)SDF-1的影響,β-Gal染色法鑒定NIH3T3/LacZ細(xì)胞。用改良Boyden小室法檢測(cè)NIH3T3/SDF-1細(xì)胞表達(dá)的SDF-1對(duì)內(nèi)皮祖細(xì)胞EPCs體外遷移作用,DAPI染色法檢測(cè)SDF-1對(duì)EPCs凋亡影響。 結(jié)果:凍存細(xì)胞可順利解凍,培養(yǎng)細(xì)胞存活細(xì)胞率≥90%。轉(zhuǎn)導(dǎo)入SDF-1表達(dá)載體的NIH 3T3/SDF-1細(xì)胞24小時(shí)可產(chǎn)生147±6 ng SDF-1/10~6細(xì)胞,轉(zhuǎn)導(dǎo)入LacZ表達(dá)載體的NIH3T3/LacZ細(xì)胞可表達(dá)LacZ,細(xì)胞β-Gal染色陽(yáng)性,為藍(lán)色。EPO的存在并不能直接影響NIH3T3和NIH3T3/SDF-1細(xì)胞中SDF-1的表達(dá)。當(dāng)EPCs與NIH3T3/SDF-1細(xì)胞共培養(yǎng)時(shí)同單獨(dú)NIH3T3細(xì)胞共培養(yǎng)時(shí)相比遷移較多,(分別為31±9和13±4遷移細(xì)胞,P＜0.05)。并且,當(dāng)在L-NMMA存在時(shí),EPCs和NIH 3T3/SDF-1細(xì)胞共培養(yǎng)后遷移細(xì)胞明顯減少為16±4(P＜0.05)。而與NIH 3T3/SDF-1細(xì)胞共培養(yǎng)的EPCs中凋亡發(fā)生率與同NIH 3T3細(xì)胞共培養(yǎng)的EPCs中凋亡發(fā)生率相比較低(分別為17±4%,30±7%P＜0.05)。L-NMMA的存在并不明顯影響與NIH 3T3/SDF-1細(xì)胞共培養(yǎng)的EPCs中凋亡發(fā)生率(18±5%P＞0.05)。 結(jié)論:體外遷移實(shí)驗(yàn)表明EPC可在NIH3T3/SDF-1細(xì)胞分泌SDF-1的趨化作用下遷移,SDF-1可抑制EPCs凋亡。SDF-1遷移效應(yīng)是通過(guò)一氧化氮(NO)途徑,但NO沒(méi)有參加SDF-1抑制EPC凋亡的過(guò)程。EPO并不影響NIH3T3/SDF-1細(xì)胞SDF-1的表達(dá)。本研究為下一步研究SDF-1在體內(nèi)趨化EPC以及促血管新生的研究打下了必要的基礎(chǔ)。 第三部分促紅細(xì)胞生成素聯(lián)合基質(zhì)細(xì)胞衍生因子對(duì)小鼠缺血下肢血管新生的作用 目的:促紅細(xì)胞生成素(EPO)可促進(jìn)骨髓單核細(xì)胞動(dòng)員到外周血中,基質(zhì)細(xì)胞衍生因子1(SDF-1)可促進(jìn)從骨髓動(dòng)員后的祖細(xì)胞的在缺血組織中的歸巢和血管新生。我們假設(shè)聯(lián)合應(yīng)用EPO和SDF-1會(huì)大大增加促進(jìn)缺血組織中血管新生的作用。 方法:將C57BL/6J小鼠股動(dòng)脈和股靜脈切除后制作小鼠下肢缺血模型,分為EPO或SDF-1單獨(dú)處理組和聯(lián)合應(yīng)用組。SDF-1應(yīng)用方法為在缺血肌肉中立即注射NIH3T3/SDF-1細(xì)胞(10~6)以局部表達(dá)SDF-1,EPO應(yīng)用方法為術(shù)后促紅細(xì)胞生成素(EPO 1000IU/kg/day)每天腹腔注射,連續(xù)3天。小鼠缺血模型下肢血流灌注情況用激光多普勒掃描測(cè)定。下肢缺血手術(shù)后21天處死小鼠并用免疫組化和Western印跡方法分析缺血肌肉組織中細(xì)胞SDF-1表達(dá),免疫方法檢測(cè)缺血組織內(nèi)CD34~+細(xì)胞,堿性磷酸酶染色方法評(píng)估毛細(xì)血管密度,原位凋亡測(cè)定試劑盒檢測(cè)肌肉細(xì)胞的凋亡。全自動(dòng)血液分析儀檢測(cè)外周血液學(xué)參數(shù)以明確EPO對(duì)外周血細(xì)胞的影響。 結(jié)果:免疫組化和Wwestern印跡分析表明注射的NIH3T3/SDF-1細(xì)胞在缺血組織內(nèi)生存并表達(dá)SDF-1。用SDF-1或EPO單獨(dú)處理3周后缺血/非缺血肢體的血流灌注比分別增加到0.75±0.06和0.63±0.03,同注射生理鹽水對(duì)照組相比增加明顯(0.50±0.02,P＜0.05)。而聯(lián)合應(yīng)用SDF-1和EPO組小鼠缺血下肢血流灌注比增加更明顯(0.89±0.08同單獨(dú)一種處理相比P＜0.05)。在SDF-1和EPO處理組,CD34~+細(xì)胞增加,毛細(xì)血管密度增加和肌肉細(xì)胞凋亡減少均比其他組相比有意義(同其他組相比P＜0.05)。外周血紅細(xì)胞數(shù)目和紅細(xì)胞壓積無(wú)明顯增加。 結(jié)論:在小鼠下肢缺血模型中聯(lián)合應(yīng)用EPO增強(qiáng)EPCs動(dòng)員和增加SDF-1調(diào)節(jié)的內(nèi)皮祖細(xì)胞(EPCs)歸巢可促進(jìn)血管新生和增加血流灌注。同時(shí),短期應(yīng)用EPO并不增加紅細(xì)胞數(shù)量和紅細(xì)胞壓積。
[Abstract]:Isolation , culture and identification of endothelial progenitor cells from the first part



Objective : To establish a culture system of EPCs by using endothelial progenitor cells ( EPCs ) , established in the laboratory , to establish a culture system of EPCs and to detect and identify the phenotype and function of the cells .



Methods : 20 ml of peripheral blood were collected from the peripheral blood of New Zealand white rabbits . The cells were cultured in a culture dish coated with 0.1 % fibronectin . The cells were cultured in a culture dish coated with 0.1 % fibronectin . The expression of CD133 , CD34 , VWF and CD45 antigens was detected on the 7th day of culture . The endothelial function was identified by DiI - acLDL and FITC - UEA - 1 absorption test .



Results : The positive rates of CD133 , CD34 and VWF were 83 . 11 鹵 3 . 46 % , 82 . 87 鹵 4 . 66 % , 77.45 鹵 6.71 % and 45 % , respectively .



Conclusion : The method is used to isolate , culture and amplify EPC from peripheral blood pathway , and has the characteristics and function of endothelial progenitor cells .



The in vitro effect of the second part of stromal cell - derived factor - 1 on endothelial progenitor cells



Objective : To prepare the virus gene vector transduced into NIH 3T3 / SDF - 1 and NIH 3T3 cells and to detect the function of cells , and to investigate the effects of its secretion matrix cell - derived factor - 1 ( SDF - 1 ) on the in vitro culture and amplification of EPC .



Methods : The effects of SDF - 1 on the expression of SDF - 1 and the presence of EPO ( 10 IU / ml ) on the expression of SDF - 1 and the presence of EPO ( 10 IU / ml ) were determined by using the method of ELASA . The effect of SDF - 1 on the expression of SDF - 1 was detected by the modified Boyden chamber method . The effect of SDF - 1 on the apoptosis of EPCs was detected by modified Boyden chamber method .



Results : The survival rate of cultured cells was 鈮，

本文編號(hào)：1831802