表达hCD4和hCCR5基因的骨髓间充质干细胞模型的建立

发布时间：2018-04-11 17:42

  本文选题：骨髓间充质干细胞 + HIV-　； 参考：《重庆医学》2017年32期

【摘要】：目的研制一种能够表达hCD4和hCCR5基因的骨髓间充质干细胞(MSCs)模型,以应用于Ⅰ型人类免疫缺陷病毒(HIV-1)研究。方法构建PLA.ExBi.P/puro-CMV-CD4-IRES-CCR慢病毒载体,按最佳感染复数转染MSCs,荧光定量PCR检测hCD4和hCCR5的mRNA的表达,免疫印迹法检测MSCs中hCD4和hCCR5蛋白的表达,免疫荧光法检测hCD4和hCCR5荧光的表达;将建立的MSCs细胞进行HIV-1感染后,PCR检测HIV RNA的表达。结果 MSCs经转染慢病毒载体后,hCD4和CCR5mRNA凝胶电泳可见193bp和75bp的阳性条带,mRNA的qRT-PCR检测可见其表达升高(P0.01);Western blot检测可见55.0×10~3(hCD4)和40.6×10~3(hCCR5)的阳性条带;免疫荧光成像可见存在CD4和CCR5的荧光表达,而空载慢病毒载体呈阴性。转染后MSCs细胞感染HIV-1后在培养细胞与上清液中HIV-1RNA均显著升高(P0.05)。结论成功建立了高效表达hCD4/CCR5蛋白的骨髓间充质干细胞模型,具备对HIV-1易感的特性,可应用于HIV的发病机制、抗病毒、疫苗开发等研究。
[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.
【作者单位】： 广东医科大学附属龙华中心医院;桂林医学院生物技术学院;中山大学生命科学院;广州中医药大学热带医学研究所;桂林医学院广西肝脏损伤与修复分子医学重点实验室;
【基金】：国家自然科学基金资助项目(81660755;81573861) 深圳市科技计划项目(JCYJ20170307160524377)
【分类号】：R512.91
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本文编号：1737010