基于小鼠白血病细胞株的HIV-1感染细胞模型研究

发布时间：2018-06-19 08:11

本文选题：AIDS/HIV-1 + 鼠白血病细胞系(L615及L1210)　；参考：《桂林医学院》2017年硕士论文

【摘要】：目的:基于鼠白血病细胞(L615和L1210),建立一种能够表达hCD4/CCR5/cyclinT1的跨物种感染HIV-1的细胞模型。人类免疫缺陷病毒(Human immunodeficiency virus,HIV)是一种感染人体免疫系统细胞,破坏和损伤其功能,最终导致获得性免疫缺陷综合征(acquired immunodeficiency syndrome,AIDS)即艾滋病发生的病原体。艾滋病自1981年第一次报道以来,作为一种公共卫生和社会问题,就一直危害着人类的生存和发展。HIV具有很强的物种特异性,这导致,在非人灵长类动物中采用嵌合的猴-人免疫缺陷病毒(simian-human immunodeficiency virus,SHIV)进行细胞与动物模型的制备;在鼠类中,仅能通过选择重症联合免疫缺陷鼠(severe combined immunodeficiency,SCID),移植人类组织细胞实现HIV-1的感染而进行动物模型的制备。为实现HIV-1跨种族于鼠细胞的HIV-1入胞感染以及病毒的完整复制的细胞模型,而设置本课题研究。方法:首先,应用Gateway技术,构建慢病毒质粒pLV[Exp]-EGFP/Neo-CMVhCD4:T2A:hCCNT1:IRES:hCCR5,经核苷酸测序分析确认目的基因无误后进行慢病毒表达载体包装;将制备的慢病毒载体感染293T细胞,采用qRT-PCR检测293T细胞中hCD4、hCCR5和hCyclinT1基因在mRNA水平的表达情况,以明确制备的慢病毒载体hCD4/CCR5/CyclinT1基因表达情况。然后,将加载hCD4/CCR5/cyclinT1的慢病毒载体以最佳感染复数感染L615及L1210细胞,提取L615及L1210的RNA和蛋白质用实时荧光定量PCR(RT-PCR)、免疫印迹法(Western blot)、免疫荧光法检测L615及L1210细胞中hCD4、hCCR5和hCyclinT1的表达率。最后,对转基因的L615及L1210进行HIV-1感染,通过RT-PCR检测细胞培养上清液及胞体中的HIV-1RNA,验证hCD4/CCR5/cyclinT1转基因小鼠细胞对HIV-1感染的敏感性。结果:核苷酸测序结果表明hCD4、hCCR5和hCyclinT1均已正确地插入到质粒载体中;通过荧光显微镜可以直接观察到由EGFP标记的慢病毒载体;qRT-PCR结果实验结果表明,与空载慢病毒相比,基于表达hCD4/CCR5/cyclinT1分子的慢病毒载体的hCD4、hCCR5和hCyclinT1基因在mRNA水平的表达量相当高,这表明基于表达hCD4/CCR5/cyclinT1分子的慢病毒载体是成功构建的。L615及L1210细胞经慢病毒载体转染后,qRT-PCR结果说明这两种细胞可以阳性表达h CD4、hCCR5和h CyclinT1mRNA;Western blot检测结果显示L615及L1210细胞中hCD4、hCCR5和hCyclinT1蛋白的表达;免疫荧光的结果表明hCD4、hCCR5和hCyclinT1是可以在L615及L1210细胞膜上表达的。以上研究结果证实,慢病毒载体可将外源基因转入细胞,通过慢病毒载体介导hCD4/CCR5/cyclinT1进入L615和L1210细胞是可行的。转基因的L615及L1210感染HIV-1后,RT-PCR结果发现在两种细胞系的培养上清液及胞体中都检测到HIV-1RNA,说明在感染表达hCD4/CCR5/cyclinT1的慢病毒后,L615及L1210可以支持HIV-1的入胞感染及在细胞内的有效病毒复制。结论:本实验建立了能够支持HIV-1入胞感染、并实现病毒复制的小鼠白血病细胞模型(L615和L1210),本研究的小鼠细胞模型还可以进一步促进HIV-1感染的鼠类动物模型研究与制备。跨物种的鼠类的HIV-1感染的细胞与动物模型,可为HIV/AIDS的发病机制、疫苗制备和潜在的抗病毒药物的研究提供一个新的平台。
[Abstract]:Objective: Based on L615 and L1210, a cross species HIV-1 cell model that can express hCD4/CCR5/cyclinT1 is established. The human immunodeficiency virus (Human immunodeficiency virus, HIV) is an infected human immune system cell that destroys and damages its function and eventually leads to acquired immunodeficiency syndrome (acquir). Ed immunodeficiency syndrome, AIDS) is the pathogen of AIDS. Since the first report of AIDS in 1981, as a public health and social problem, it has been harmful to the survival and development of human.HIV with a strong species-specificity, which leads to the use of chimeric monkey human immunodeficiency disease in non human primates. Simian-human immunodeficiency virus (SHIV) is used for the preparation of cell and animal models; in rats, only by selecting severe combined immunodeficiency mice (severe combined immunodeficiency, SCID), human tissue cells are transplanted to achieve HIV-1 infection and into the preparation of an action model. To realize HIV-1 trans race to mouse cells HIV-1 Cell model of infective infection and complete replication of the virus, and set up this topic. Methods: first, Gateway technology was used to construct the lentivirus plasmid pLV[Exp]-EGFP/Neo-CMVhCD4:T2A:hCCNT1:IRES:hCCR5, and the lentivirus expression vector was packed after the nucleotide sequencing analysis confirmed that the target gene was unmistakable, and the lentivirus vector was prepared. 293T cells were stained with qRT-PCR to detect the expression of hCD4, hCCR5 and hCyclinT1 genes at mRNA level in 293T cells in order to identify the hCD4/CCR5/CyclinT1 gene expression of the lentivirus vector. Then, the lentivirus vector loaded with hCD4/CCR5/cyclinT1 was loaded to the L615 and L1210 cells with the best number of infection. The expression of hCD4, hCCR5 and hCyclinT1 in L615 and L1210 cells was detected by real time fluorescence quantitative PCR (RT-PCR) and immunoblotting (Western blot), and the expression rate of hCD4, hCCR5 and hCyclinT1 in L615 and L1210 cells was detected by immunofluorescence. Finally, the HIV-1 infection was carried out on the L615 and L1210 of the transgenic cells. The sensitivity of mouse cells to HIV-1 infection. Results: nucleotide sequencing results showed that hCD4, hCCR5 and hCyclinT1 were correctly inserted into plasmid vectors; EGFP labeled lentivirus carriers could be directly observed by fluorescence microscopy; the results of qRT-PCR results showed that the expression of hCD4/CCR5/cyclinT1 points was compared with the no-load lentivirus. The expression of hCD4, hCCR5 and hCyclinT1 genes at the mRNA level of the lentivirus vector is quite high, which indicates that the lentivirus vector based on the expression of hCD4/CCR5/cyclinT1 molecule is a successful construction of.L615 and L1210 cells transfected by the lentivirus vector. The result of qRT-PCR shows that the two cells can positive expression h CD4, hCCR5 and H. The results of tern blot detection showed the expression of hCD4, hCCR5 and hCyclinT1 proteins in L615 and L1210 cells, and the results of immunofluorescence showed that hCD4, hCCR5 and hCyclinT1 could be expressed on L615 and L1210 cell membranes. The results of the study confirmed that the lentivirus vector could transfer the exogenous gene into the cell and mediate the expression through the lentivirus vector. It is feasible to enter L615 and L1210 cells. After transgene L615 and L1210 infected HIV-1, RT-PCR results found that HIV-1RNA was detected in the culture supernatant and cell of two cell lines, indicating that L615 and L1210 can support HIV-1 cell infection and effective virus replication in cells after the infection of the lentivirus expressing hCD4/CCR5/cyclinT1. Conclusion: this experiment established a mouse leukemia cell model (L615 and L1210) that can support HIV-1 infection and the replication of the virus. The mouse model of this study can further promote the study and preparation of rat model of HIV-1 infected rats. The cell and animal model of the HIV-1 infection of the rodent species across species can be HIV/AIDS The pathogenesis, vaccine preparation and potential antiviral drug research will provide a new platform.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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