發(fā)布時(shí)間：2019-03-19 09:56

【摘要】： 目的：探索人臍帶血血管內(nèi)皮祖細(xì)胞(EPCs)分離、培養(yǎng)、擴(kuò)增和鑒定的方法；探討HIV-1來源的慢病毒載體介導(dǎo)綠色熒光蛋白(GFP)基因轉(zhuǎn)染血管內(nèi)皮祖細(xì)胞的可行性和方法。方法：采用密度梯度離心法和貼壁細(xì)胞分離法相結(jié)合,分離和篩選出人臍帶血內(nèi)皮祖細(xì)胞,用EGM-2培養(yǎng)基培養(yǎng)、擴(kuò)增,采用細(xì)胞免疫熒光染色和流式細(xì)胞儀檢測(cè)內(nèi)皮祖細(xì)胞表面標(biāo)記CD133、CD34、VEGFR-2的方法鑒定實(shí)驗(yàn)培養(yǎng)的內(nèi)皮祖細(xì)胞。取生長活躍的EPCs,以HIV-1來源的慢病毒為載體,以綠色熒光蛋白(GFP)基因?yàn)槟康幕蜣D(zhuǎn)染EPCs。感染復(fù)數(shù)MOI分別為：1：10,1：50,1：100,MTT法檢測(cè)不同病毒滴度時(shí)細(xì)胞增殖情況,計(jì)算轉(zhuǎn)染率；取1：50轉(zhuǎn)染組與未轉(zhuǎn)染的空白組進(jìn)行對(duì)照,于轉(zhuǎn)染后1-10天觀察兩組細(xì)胞增殖情況。用t檢驗(yàn)、卡方檢驗(yàn)、方差分析等方法對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果：單個(gè)核細(xì)胞經(jīng)EGM-2培養(yǎng)基培養(yǎng)一周后,即分化成CD133、KDR和CD34陽性的EPCs。1:10轉(zhuǎn)染和1：50轉(zhuǎn)染后細(xì)胞增殖無明顯影響,細(xì)胞顯綠色熒光。1：10比率轉(zhuǎn)染后48小時(shí)綠色熒光細(xì)胞比率僅(35.2±2.75)%；1：50比率轉(zhuǎn)染后48小時(shí)熒光顯微鏡下觀察轉(zhuǎn)染率為(87.4±1.89)%,轉(zhuǎn)染率高于1：10組(P0.05)。1：50轉(zhuǎn)染后的細(xì)胞繼續(xù)培養(yǎng)并與未轉(zhuǎn)染組對(duì)照,顯示兩組細(xì)胞生長曲線無明顯差異(P0.05)。1：100比率轉(zhuǎn)染后,細(xì)胞的增殖處于停滯狀態(tài),鏡下可見細(xì)胞壞死形成碎片。結(jié)論：1、采用密度梯度離心法和貼壁細(xì)胞分離法相結(jié)合,經(jīng)EGM-2培養(yǎng)基培養(yǎng)擴(kuò)增可從臍帶血獲得較高純度和較大數(shù)量的EPCs;2、采用HIV-1來源的慢病毒載體介導(dǎo)綠色熒光蛋白基因轉(zhuǎn)染標(biāo)記血管內(nèi)皮祖細(xì)胞是可行的；以MOI=1：50進(jìn)行轉(zhuǎn)染,對(duì)細(xì)胞生長影響小,轉(zhuǎn)染效率高。
[Abstract]:Objective: To explore the methods of isolation, culture, amplification and identification of human umbilical cord blood vessel endothelial progenitor cells (EPCs), and to explore the feasibility and method of the lentiviral vector-mediated green fluorescent protein (GFP) gene from HIV-1. Methods: The human umbilical cord blood endothelial progenitor cells were isolated and screened by density gradient centrifugation and adherent cell separation. The endothelial progenitor cells were cultured and amplified with EGM-2 medium, and the surface markers of the endothelial progenitor cells were detected by immunofluorescence staining and flow cytometry. VEGFR-2 was used to identify the cultured endothelial progenitor cells. EPCs were transfected with the lentivirus of HIV-1, and the EPCs were transfected with the green fluorescent protein (GFP) gene as the target gene. The proliferation of cells was detected by MTT method in 1:10,1:50,1:100 and MTT method, and the proliferation of two groups was observed 1-10 days after transfection. The results of the experiment were statistically analyzed by t-test, chi-square test and variance analysis. Results: One week after the single core cell was cultured with EGM-2 medium, the cells were differentiated into CD133, KDR and CD34-positive EPCs.1:10 was transfected and the cell proliferation was not significantly affected after 1:50 transfection. The ratio of green fluorescent cells was only 35.2 (2.75)% after 1:10 ratio transfection. The transfection rate was 87.4% 1.89% and the transfection rate was higher than that of 1:10 (P0.05). The proliferation of the cells was in a dead state, and the cells were seen to form fragments under the microscope. Conclusion:1. High-purity and large number of EPCs can be obtained from umbilical cord blood by the combination of density gradient centrifugation and adherent cell separation. Transfection of the green fluorescent protein gene with a lentiviral vector from the HIV-1 source is feasible; the transfection is performed with MOI = 1:50, the effect on the growth of the cells is small, and the transfection efficiency is high.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李傳勇;李曉強(qiáng);姜坤;孟慶友;于小濱;雷鋒銳;;綠色熒光蛋白基因體外轉(zhuǎn)染骨髓源性血管內(nèi)皮祖細(xì)胞[J];中國組織工程研究與臨床康復(fù);2009年14期

，

本文編號(hào)：2443419