本文選題：AIDS/HIV-1 + 鼠白血病細(xì)胞系(L615及L1210)　； 參考：《桂林醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:基于鼠白血病細(xì)胞(L615和L1210),建立一種能夠表達(dá)hCD4/CCR5/cyclinT1的跨物種感染HIV-1的細(xì)胞模型。人類免疫缺陷病毒(Human immunodeficiency virus,HIV)是一種感染人體免疫系統(tǒng)細(xì)胞,破壞和損傷其功能,最終導(dǎo)致獲得性免疫缺陷綜合征(acquired immunodeficiency syndrome,AIDS)即艾滋病發(fā)生的病原體。艾滋病自1981年第一次報道以來,作為一種公共衛(wèi)生和社會問題,就一直危害著人類的生存和發(fā)展。HIV具有很強(qiáng)的物種特異性,這導(dǎo)致,在非人靈長類動物中采用嵌合的猴-人免疫缺陷病毒(simian-human immunodeficiency virus,SHIV)進(jìn)行細(xì)胞與動物模型的制備;在鼠類中,僅能通過選擇重癥聯(lián)合免疫缺陷鼠(severe combined immunodeficiency,SCID),移植人類組織細(xì)胞實(shí)現(xiàn)HIV-1的感染而進(jìn)行動物模型的制備。為實(shí)現(xiàn)HIV-1跨種族于鼠細(xì)胞的HIV-1入胞感染以及病毒的完整復(fù)制的細(xì)胞模型,而設(shè)置本課題研究。方法:首先,應(yīng)用Gateway技術(shù),構(gòu)建慢病毒質(zhì)粒pLV[Exp]-EGFP/Neo-CMVhCD4:T2A:hCCNT1:IRES:hCCR5,經(jīng)核苷酸測序分析確認(rèn)目的基因無誤后進(jìn)行慢病毒表達(dá)載體包裝;將制備的慢病毒載體感染293T細(xì)胞,采用qRT-PCR檢測293T細(xì)胞中hCD4、hCCR5和hCyclinT1基因在mRNA水平的表達(dá)情況,以明確制備的慢病毒載體hCD4/CCR5/CyclinT1基因表達(dá)情況。然后,將加載hCD4/CCR5/cyclinT1的慢病毒載體以最佳感染復(fù)數(shù)感染L615及L1210細(xì)胞,提取L615及L1210的RNA和蛋白質(zhì)用實(shí)時熒光定量PCR(RT-PCR)、免疫印跡法(Western blot)、免疫熒光法檢測L615及L1210細(xì)胞中hCD4、hCCR5和hCyclinT1的表達(dá)率。最后,對轉(zhuǎn)基因的L615及L1210進(jìn)行HIV-1感染,通過RT-PCR檢測細(xì)胞培養(yǎng)上清液及胞體中的HIV-1RNA,驗(yàn)證hCD4/CCR5/cyclinT1轉(zhuǎn)基因小鼠細(xì)胞對HIV-1感染的敏感性。結(jié)果:核苷酸測序結(jié)果表明hCD4、hCCR5和hCyclinT1均已正確地插入到質(zhì)粒載體中;通過熒光顯微鏡可以直接觀察到由EGFP標(biāo)記的慢病毒載體;qRT-PCR結(jié)果實(shí)驗(yàn)結(jié)果表明,與空載慢病毒相比,基于表達(dá)hCD4/CCR5/cyclinT1分子的慢病毒載體的hCD4、hCCR5和hCyclinT1基因在mRNA水平的表達(dá)量相當(dāng)高,這表明基于表達(dá)hCD4/CCR5/cyclinT1分子的慢病毒載體是成功構(gòu)建的。L615及L1210細(xì)胞經(jīng)慢病毒載體轉(zhuǎn)染后,qRT-PCR結(jié)果說明這兩種細(xì)胞可以陽性表達(dá)h CD4、hCCR5和h CyclinT1mRNA;Western blot檢測結(jié)果顯示L615及L1210細(xì)胞中hCD4、hCCR5和hCyclinT1蛋白的表達(dá);免疫熒光的結(jié)果表明hCD4、hCCR5和hCyclinT1是可以在L615及L1210細(xì)胞膜上表達(dá)的。以上研究結(jié)果證實(shí),慢病毒載體可將外源基因轉(zhuǎn)入細(xì)胞,通過慢病毒載體介導(dǎo)hCD4/CCR5/cyclinT1進(jìn)入L615和L1210細(xì)胞是可行的。轉(zhuǎn)基因的L615及L1210感染HIV-1后,RT-PCR結(jié)果發(fā)現(xiàn)在兩種細(xì)胞系的培養(yǎng)上清液及胞體中都檢測到HIV-1RNA,說明在感染表達(dá)hCD4/CCR5/cyclinT1的慢病毒后,L615及L1210可以支持HIV-1的入胞感染及在細(xì)胞內(nèi)的有效病毒復(fù)制。結(jié)論:本實(shí)驗(yàn)建立了能夠支持HIV-1入胞感染、并實(shí)現(xiàn)病毒復(fù)制的小鼠白血病細(xì)胞模型(L615和L1210),本研究的小鼠細(xì)胞模型還可以進(jìn)一步促進(jìn)HIV-1感染的鼠類動物模型研究與制備。跨物種的鼠類的HIV-1感染的細(xì)胞與動物模型,可為HIV/AIDS的發(fā)病機(jī)制、疫苗制備和潛在的抗病毒藥物的研究提供一個新的平臺。
[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.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91

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