本文選題：骨髓間充質(zhì)干細(xì)胞 + HIV-��； 參考：《重慶醫(yī)學(xué)》2017年32期

【摘要】：目的研制一種能夠表達(dá)hCD4和hCCR5基因的骨髓間充質(zhì)干細(xì)胞(MSCs)模型,以應(yīng)用于Ⅰ型人類免疫缺陷病毒(HIV-1)研究。方法構(gòu)建PLA.ExBi.P/puro-CMV-CD4-IRES-CCR慢病毒載體,按最佳感染復(fù)數(shù)轉(zhuǎn)染MSCs,熒光定量PCR檢測hCD4和hCCR5的mRNA的表達(dá),免疫印跡法檢測MSCs中hCD4和hCCR5蛋白的表達(dá),免疫熒光法檢測hCD4和hCCR5熒光的表達(dá);將建立的MSCs細(xì)胞進(jìn)行HIV-1感染后,PCR檢測HIV RNA的表達(dá)。結(jié)果 MSCs經(jīng)轉(zhuǎn)染慢病毒載體后,hCD4和CCR5mRNA凝膠電泳可見193bp和75bp的陽性條帶,mRNA的qRT-PCR檢測可見其表達(dá)升高(P0.01);Western blot檢測可見55.0×10~3(hCD4)和40.6×10~3(hCCR5)的陽性條帶;免疫熒光成像可見存在CD4和CCR5的熒光表達(dá),而空載慢病毒載體呈陰性。轉(zhuǎn)染后MSCs細(xì)胞感染HIV-1后在培養(yǎng)細(xì)胞與上清液中HIV-1RNA均顯著升高(P0.05)。結(jié)論成功建立了高效表達(dá)hCD4/CCR5蛋白的骨髓間充質(zhì)干細(xì)胞模型,具備對(duì)HIV-1易感的特性,可應(yīng)用于HIV的發(fā)病機(jī)制、抗病毒、疫苗開發(fā)等研究。
[Abstract]:Objective to develop a bone marrow mesenchymal stem cell (MSCs) model expressing hCD4 and hCCR5 genes for human immunodeficiency virus type I (HIV-1).Methods PLA.ExBi.P/puro-CMV-CD4-IRES-CCR lentivirus vector was constructed and transfected into MSCs according to the optimal number of infection. The expression of mRNA in hCD4 and hCCR5 was detected by fluorescence quantitative PCR, the expression of hCD4 and hCCR5 protein in MSCs was detected by Western blot, and the fluorescence expression of hCD4 and hCCR5 was detected by immunofluorescence.The expression of HIV RNA was detected by PCR after HIV-1 infection in the established MSCs cells.But no-loaded lentivirus vector was negative.After transfection of MSCs cells infected with HIV-1, HIV-1RNA in culture cells and supernatants increased significantly (P 0.05).Conclusion the bone marrow mesenchymal stem cell model with high expression of hCD4/CCR5 protein has been successfully established, which has the characteristics of susceptibility to HIV-1, and can be used in the study of pathogenesis, antivirus and vaccine development of HIV.
【作者單位】： 廣東醫(yī)科大學(xué)附屬龍華中心醫(yī)院;桂林醫(yī)學(xué)院生物技術(shù)學(xué)院;中山大學(xué)生命科學(xué)院;廣州中醫(yī)藥大學(xué)熱帶醫(yī)學(xué)研究所;桂林醫(yī)學(xué)院廣西肝臟損傷與修復(fù)分子醫(yī)學(xué)重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(81660755;81573861) 深圳市科技計(jì)劃項(xiàng)目(JCYJ20170307160524377)
【分類號(hào)】：R512.91
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本文編號(hào)：1737010