本文選題：人類免疫缺陷病毒(HIV) + 艾滋病��； 參考：《復(fù)旦大學(xué)》2008年碩士論文

【摘要】： 隨著人類免疫缺陷病毒(HIV)感染人數(shù)的增加,艾滋病的流行已經(jīng)給國(guó)家經(jīng)濟(jì)和社會(huì)帶來了巨大負(fù)擔(dān)。但目前的抗病毒藥物無(wú)法給患者提供徹底的治療,因此更多的研究開始轉(zhuǎn)變以往的治療思路,探索新的更有效更經(jīng)濟(jì)的艾滋病治療方法。RNAi(RNA interference,RNA干擾)研究的出現(xiàn),為HIV的抑制及治療提供了一種新的嘗試。RNAi干擾效應(yīng)是指雙鏈RNA在生物細(xì)胞內(nèi)對(duì)序列特異性基因轉(zhuǎn)錄和表達(dá)的抑制作用。RNAi的研究為控制HIV感染帶來希望,通過SiRNA對(duì)病毒生活周期的多個(gè)水平作用,可顯著抑制病毒的復(fù)制。目前已有研究報(bào)道了通過對(duì)HIV-1 gag,tat,rev和宿主CD4及CCR5受體進(jìn)行RNA干擾,進(jìn)而達(dá)到抑制HIV病毒復(fù)制的目的,并且研究取得了一定的進(jìn)展,為抗HIV治療開辟了一條新路。 HIV-1的特點(diǎn)之一是具有多個(gè)調(diào)節(jié)基因,這些基因可編碼相應(yīng)的調(diào)控蛋白,如Nef、Tat、Rev、Vif、Vpr、Vpx、Vpu等,它們?cè)赗NA轉(zhuǎn)錄、轉(zhuǎn)錄后加工、蛋白質(zhì)翻譯直到病毒顆粒從細(xì)胞膜釋放的過程中發(fā)揮重要的調(diào)節(jié)作用。Vif蛋白被歸于附屬蛋白質(zhì),編碼一個(gè)含有192個(gè)氨基酸,相對(duì)分子質(zhì)量大約23000的胞質(zhì)蛋白,vif基因比HIV-1基因組中env、gag等突變率高的基因相對(duì)保守。但10多年前就已經(jīng)知道Vif是HIV-1感染所必須的調(diào)控蛋白,vif基因缺陷性(△vif)的HIV-1和SIV在體內(nèi)無(wú)法復(fù)制,在體外雖可正常復(fù)制并產(chǎn)生病毒顆粒但其感染性比野生型降低近1000倍以上。vif缺陷性的病毒顆粒在形態(tài)學(xué)上也表現(xiàn)異常。在子彈頭狀病毒核心的電子密集物質(zhì)明顯減少,而核心與包膜之間的空間里電子密集物質(zhì)明顯增加,使核心形狀呈多樣化。另外,在感染細(xì)胞中的反轉(zhuǎn)錄過程以及病毒顆粒中的內(nèi)源性反轉(zhuǎn)錄也是明顯受損,因而不能感染靶細(xì)胞。有趣的是,vif缺陷性的病毒在一些腫瘤細(xì)胞系可以順利的進(jìn)行復(fù)制。根據(jù)△vif HIV-1是否能夠在其內(nèi)復(fù)制可將細(xì)胞分為“允許細(xì)胞”和“非允許細(xì)胞”�！鱲if HIV-1病毒可以進(jìn)入非允許細(xì)胞中,但在反轉(zhuǎn)錄過程中或反轉(zhuǎn)錄之后到整合之前由于某種尚不清楚的機(jī)制導(dǎo)致感染終止。早期研究發(fā)現(xiàn),除了感染性降低1000倍以上外,△vif HIV-1同野生型HIV-1相比較,二者在RNA結(jié)構(gòu)及蛋白表達(dá)等方面均未發(fā)現(xiàn)任何差別。近期由于載脂蛋白B mRNA編輯酶催化多肽樣蛋白3G(apolipoprotein B mRNAediting enzyme catalytic polypeptide like 3G,APOBEC3G)的發(fā)現(xiàn)以及對(duì)APOBEC3G與Vif相互作用機(jī)制的深入研究,使越來越多的研究者開始重視vif基因及其表達(dá)產(chǎn)物在HIV生命周期中的作用。 經(jīng)過科研人員數(shù)年的努力,現(xiàn)在RNA干擾的機(jī)制已經(jīng)得到比較清楚的闡述。RNAi是一種進(jìn)化上保守的抵御轉(zhuǎn)基因或外來病毒侵犯的防御機(jī)制,是指內(nèi)源性或外源性大約為21～23bp的核苷酸與靶基因的轉(zhuǎn)錄產(chǎn)物mRNA存在同源互補(bǔ)序列的雙鏈RNA(double-stranded RNA,dsRNA)在細(xì)胞內(nèi)特異地降解該mRNA,從而致使特異性的基因有效封閉的過程,是一種序列特異性的轉(zhuǎn)錄后基因沉默(post-transcriptional gene silencing,PTGS)。RNAi作為一種簡(jiǎn)單、快速、特異、高效、經(jīng)濟(jì)、效果可預(yù)測(cè)的技術(shù),具有明顯的優(yōu)點(diǎn),它比反義核酸技術(shù)優(yōu)越,比基因敲除簡(jiǎn)單,具有很好的應(yīng)用前景。具體可用于基因功能分析、研究信號(hào)傳導(dǎo)通路的新途徑、新藥物的研究和開發(fā)、基因治療、病毒感染治療以及腫瘤治療等各個(gè)方面。作為一種新的基因治療藥物,SiRNA可作為已有抗HIV病毒藥物的輔助治療,與其它抗病毒藥物不會(huì)互相產(chǎn)生干擾,效果更好,相信隨著對(duì)RNAi機(jī)制的進(jìn)一步認(rèn)識(shí),這一技術(shù)能夠成為抗病毒,特別是抗HIV感染的有力武器。因此本研究選擇HIV-1 vif基因作為RNAi的靶目標(biāo),通過設(shè)計(jì)針對(duì)vif的特異性SiRNA對(duì)HIV病毒RNA進(jìn)行干擾降解,進(jìn)而達(dá)到降低或抑制病毒復(fù)制的目的。研究中我們成功的設(shè)計(jì)了3段vif特異性SiRNA,對(duì)轉(zhuǎn)接入pEGFP-N1質(zhì)粒中的vif基因進(jìn)行干擾,并分別在核酸和蛋白水平進(jìn)行了驗(yàn)證,結(jié)果證明了RNAi可以有效且特異性的下調(diào)Vif蛋白的表達(dá)。 本課題的研究目的就是利用RNAi技術(shù)對(duì)HIV-1 vif基因的轉(zhuǎn)錄水平進(jìn)行下調(diào),達(dá)到抑制Vif蛋白表達(dá)的目的,為新的抗HIV治療和預(yù)防研究提供理論基礎(chǔ)。研究首先選擇人胚腎293T(HEK 293T)細(xì)胞作為宿主細(xì)胞,EGFP融合蛋白作為報(bào)告基因,觀察HIV-1 Vif蛋白在細(xì)胞內(nèi)的表達(dá)情況,為后續(xù)的RNAi做好準(zhǔn)備。第一部分先建立pEGFP-N1-vif融合質(zhì)粒,進(jìn)行HEK 293T細(xì)胞轉(zhuǎn)染,熒光顯微鏡觀察轉(zhuǎn)染細(xì)胞中的轉(zhuǎn)染效率,蛋白印跡免疫分析技術(shù)(Western blot)檢測(cè)Vif蛋白的表達(dá)情況。結(jié)果顯示,HIV-1 Vif可以在HEK 293T細(xì)胞中成功表達(dá)。采用體外轉(zhuǎn)錄法合成SiRNA,首先合成六段SiRNA(四段針對(duì)vif和一段陽(yáng)性對(duì)照、一段陰性對(duì)照)的正反義鏈模板,體外進(jìn)行反轉(zhuǎn)錄,得到長(zhǎng)21bp的雙鏈SiRNA,然后雙鏈SiRNA分別與pEGFP-N1-vif融合質(zhì)粒共轉(zhuǎn)染HEK 293T細(xì)胞。熒光下觀察綠色熒光,篩選出能抑制HIV-1 vif基因表達(dá)的有效SiRNA。驗(yàn)證轉(zhuǎn)染成功后進(jìn)行細(xì)胞抽提總RNA,進(jìn)行實(shí)時(shí)定量RT-PCR(Real-time PCR),驗(yàn)證干擾效果,Western blot檢測(cè)蛋白表達(dá)情況。結(jié)果顯示,與陰性對(duì)照比較,有3條SiRNA可明顯抑制vifmRNA的表達(dá),S-SiRNA1、S-SiRNA2組和S-SiRNA3分別下降了約53%、60%和63%;同時(shí)結(jié)果顯示蛋白表達(dá)水平也明顯降低,3條SiRNA的蛋白條帶均明顯減弱。最終驗(yàn)證了RNAi可以對(duì)HIV-1 vif進(jìn)行有效且特異性的下調(diào)表達(dá)。 本實(shí)驗(yàn)成功的建立了哺乳動(dòng)物細(xì)胞pEGFP-N1-HIV-1/vif表達(dá)系統(tǒng),并得以驗(yàn)證,Vif蛋白可以在體外哺乳動(dòng)物細(xì)胞中高水平表達(dá)。研究應(yīng)用RNAi技術(shù),對(duì)HIV-1 vif從轉(zhuǎn)錄和表達(dá)水平進(jìn)行了沉默,并分別在核酸和蛋白水平進(jìn)行了驗(yàn)證,這對(duì)于阻斷病毒和宿主細(xì)胞結(jié)合及相互作用有重要啟示,表明vif可以作為進(jìn)一步研究抗HIV-1的潛力靶點(diǎn),也為這一基因水平治療手段的開展進(jìn)行了積累。同時(shí)證實(shí),RNAi作為一種簡(jiǎn)單、快速、特異、高效、經(jīng)濟(jì)、效果可預(yù)測(cè)的技術(shù),具有明顯的優(yōu)點(diǎn)。但值得注意的是,本實(shí)驗(yàn)中的SiRNA也不能實(shí)現(xiàn)對(duì)HIV-1 vif的徹底敲除,干擾效率維持在50%左右。曾有研究報(bào)道,RNAi對(duì)HIV-1的干擾效率可以達(dá)到80%～95%左右。本實(shí)驗(yàn)考慮可能與實(shí)驗(yàn)操作和技術(shù)有一定的關(guān)系,也可能是vif基因的RNAi干擾效率較低造成的。但總之,RNAi研究的出現(xiàn),為HIV的抑制及治療提供了一種新的嘗試。 RNAi技術(shù)的問世是生命科學(xué)發(fā)展史上的一大豐碑,若能長(zhǎng)期穩(wěn)定應(yīng)用于抗病毒治療,尤其是抗HIV治療,無(wú)疑將造福人類。雖然目前RNAi技術(shù)在抗病毒的實(shí)驗(yàn)研究方面已取得可喜成績(jī),但由于諸多問題的存在,其應(yīng)用于臨床還有一段艱難漫長(zhǎng)的道路。從理論和技術(shù)兩個(gè)層面來解決RNAi的分子基礎(chǔ)及其治療應(yīng)用問題任重而道遠(yuǎn),因此對(duì)此項(xiàng)技術(shù)還需進(jìn)一步的改進(jìn)和創(chuàng)新。目前最好的艾滋病治療方法,是用多種藥物來干擾艾滋病毒生命周期中兩種重要物質(zhì)—反轉(zhuǎn)錄酶和蛋白酶—的作用,融合抑制劑與整合酶抑制劑也已成功應(yīng)用于臨床。但該方法并不能完全清除病毒,而且部分病人用藥一段時(shí)間后,體內(nèi)病毒產(chǎn)生了耐藥性。因此,從HIV病毒生命周期的其它角度著手,開發(fā)新型藥物,就顯得十分重要。有研究者預(yù)期,新型療法或許可以與現(xiàn)行的反轉(zhuǎn)錄酶抑制劑和蛋白酶抑制劑聯(lián)合使用,適用于所有艾滋病毒感染者,而RNAi就是一種新的嘗試和新的策略。
[Abstract]:With the increase of the number of human immunodeficiency virus (HIV) infection, the epidemic of AIDS has brought a huge burden to the economy and society. But the current antiviral drugs can not provide thorough treatment to the patients, so more research begins to change the previous treatment ideas and explore new and more efficient and more economical AIDS treatment. The emergence of the study of.RNAi (RNA interference, RNA interference) provides a new attempt on the inhibition and treatment of HIV for the inhibitory effect of.RNAi on the inhibitory effect of the double chain RNA on the transcription and expression of sequence specific genes in biological cells, which brings hope for controlling HIV infection, and the multiple levels of the life cycle of the virus through SiRNA. The role of the virus can significantly inhibit the replication of the virus. At present, it has been reported that through RNA interference on HIV-1 gag, tat, rev and host CD4 and CCR5 receptors, the purpose of inhibiting the replication of HIV virus is achieved, and some progress has been made in the study, which opens a new way for the anti HIV treatment.
One of the characteristics of HIV-1 is that there are multiple regulatory genes that can encode the corresponding regulatory proteins, such as Nef, Tat, Rev, Vif, Vpr, Vpx, Vpu and so on. They play an important regulatory role in RNA transcriptional, post transcriptional processing, protein translation until the release of virus particles from the cell membrane, and the.Vif protein is assigned to the accessory protein and encodes a protein. The vif gene, which contains 192 amino acids and relative molecular mass of about 23000, is relatively conservative than the genes with high mutation rates such as Env and gag in the HIV-1 genome. But more than 10 years ago, it has been known that Vif is a necessary regulatory protein for HIV-1 infection. The HIV-1 and SIV of the vif gene defect (delta VIF) can not be replicated in the body, although it can be recovered in vitro. The virus particles that produce and produce virus particles, but the virus particles whose infectivity is nearly 1000 times more than that of the wild type, are also abnormal in morphology. The electron dense substance in the core of the bullet head virus is obviously reduced and the electron dense substance in the space between the core and the envelope is significantly increased and the core shape is diversified. In addition, the.Vif virus core is varied. The reverse transcriptional process in the infected cells and the endogenous reverse transcription in the virus particles are also significantly impaired, and therefore cannot infect the target cells. Interestingly, the Vif defective virus can be successfully replicated in some tumor cell lines. If the Vif HIV-1 can be reproduced within it, the cells can be divided into "permissive" and "non" Allow cells ". Delta Vif HIV-1 virus can enter non permissible cells, but in the reverse transcription process or after reverse transcription to integration due to some unknown mechanism leading to infection termination. Early studies found that, in addition to 1000 times the infection reduction to the upper, delta Vif HIV-1 is compared with the wild type HIV-1, the two in RNA structure and protein In recent years, the discovery of the apolipoprotein B mRNA editing enzyme catalyzed polypeptide like protein 3G (apolipoprotein B mRNAediting enzyme catalytic polypeptide like 3G, APOBEC3G) and the in-depth study of the interaction mechanism between them and the interaction mechanism have made more and more researchers begin to attach importance to the genes and their genes. The role of the expression product in the HIV life cycle.
After several years of efforts by researchers, the mechanism of RNA interference has been clearly stated that.RNAi is an evolutionary conservative defense mechanism to resist transgene or alien virus invasion, which refers to the dual chain RNA of endogenous or exogenous nucleotide and target gene transfer product mRNA with a homologous complementary sequence of 21 to 23bp. (double-stranded RNA, dsRNA) is a simple, rapid, specific, efficient, efficient, efficient, efficient, economical, and predictable technique, which is a simple, rapid, specific, efficient, efficient, cost-effective, and predictable technique, which degrade the mRNA in the cell specifically, and causes the effective closure of specific genes. It is a sequence specific post transcriptional gene silencing (post-transcriptional gene silencing, PTGS).RNAi as a simple, rapid, specific, efficient, economical and predictable technique. It is superior to antisense nucleic acid technology and is more simple than gene knockout. It can be used in gene functional analysis, new pathway of signal transduction pathway, research and development of new drugs, gene therapy, virus infection treatment and tumor treatment. As a new gene therapy drug, SiRNA can be used as a new gene therapy drug. It is believed that this technology can become a powerful weapon for anti viral, especially anti HIV infection, as the adjuvant therapy of anti HIV virus drugs and other antiviral drugs do not interfere with each other. It is believed that with the further understanding of the RNAi mechanism, this technique can be a powerful weapon for anti viral, especially anti HIV infection. Therefore, this study chose the HIV-1 vif gene as the target of RNAi and designed for vi. F specific SiRNA interferes with HIV virus RNA, and then reduces or inhibits virus replication. In the study, we successfully designed 3 segments of Vif specific SiRNA to interfere with Vif genes transferred into pEGFP-N1 plasmids, and verify the nucleic acid and protein level respectively. The results show that RNAi can be effective and specific. The expression of Vif protein is downregulated by sex.
The purpose of this study is to reduce the transcriptional level of the HIV-1 vif gene by using RNAi technology to inhibit the expression of Vif protein, and to provide a theoretical basis for the new anti HIV treatment and prevention research. First, the study selected human embryonic kidney 293T (HEK 293T) cells as host cell, EGFP fusion protein as a reporter gene, and observed HIV-1 Vi. The expression of F protein in the cell was prepared for the subsequent RNAi. The first part first established the pEGFP-N1-vif fusion plasmid, transfected HEK 293T cells, and observed the transfection efficiency in the transfected cells by fluorescence microscopy. The expression of Vif protein was detected by Western blot immunoassay (Western blot). The results showed that HIV-1 Vif could be in HEK. 293T cells were successfully expressed. SiRNA was synthesized by in vitro transcription. First, six segments of SiRNA (four segments for Vif and a positive control, a negative control) were synthesized by reverse transcription, and a long 21bp double chain SiRNA was obtained in vitro. Then the double stranded SiRNA and pEGFP-N1-vif fusion plasmid co transfected HEK 293T cells. Detection of green fluorescence, screening effective SiRNA. to inhibit the expression of HIV-1 vif gene to verify the successful transfection of the total RNA, real-time quantitative RT-PCR (Real-time PCR), verify the interference effect, Western blot detection protein expression. The results show that, compared with negative control, 3 SiRNA can obviously inhibit vifmRNA expression, S-SiRNA1 The S-SiRNA2 group and S-SiRNA3 decreased by about 53%, 60% and 63%, respectively, and the results showed that the protein expression level was also significantly reduced, and the protein bands of 3 SiRNA were obviously weakened. Finally, it was proved that RNAi could be effectively and specifically down expression of HIV-1 Vif.
This experiment successfully established the pEGFP-N1-HIV-1/vif expression system of mammalian cells and proved that Vif protein could be expressed at high level in mammalian cells in vitro. RNAi technology was used to study the transcription and expression level of HIV-1 Vif, and the level of nucleic acid and protein was verified respectively, which was used to block the virus. The binding and interaction of host cells have important implications, indicating that Vif can be used as a potential target for further research on the potential of anti HIV-1 and also for the development of this gene level treatment. It is also proved that RNAi has obvious advantages as a simple, rapid, specific, efficient, economical and predictable Technology. In this experiment, SiRNA can not complete the complete knockout of HIV-1 Vif, and the interference efficiency is about 50%. The interference efficiency of RNAi to HIV-1 can reach about 80% to 95%. This experiment may have a certain relationship with the experimental operation and technology, and can also be caused by the low RNAi interference efficiency of the vif gene. In conclusion, the emergence of RNAi provides a new attempt for inhibition and treatment of HIV.
The advent of RNAi technology is a great monument in the history of life science. If it can be applied to antiviral therapy for a long time, especially anti HIV treatment, it will undoubtedly benefit mankind. Although RNAi technology has made gratifying achievements in the experimental research of antivirus, it has a long and difficult application in clinical due to the existence of many problems. The best way to treat RNAi is to interfere with the two important substances in the life cycle of HIV - reverse transcriptase and protease in the life cycle of HIV. The effects, fusion inhibitors and integrase inhibitors have also been successfully applied to the clinic. But this method does not completely remove the virus, and some patients have a drug resistance after a period of drug use. Therefore, starting new drugs from the other angles of the HIV virus life cycle is very important. The new therapy may be combined with the current reverse transcriptase inhibitors and protease inhibitors, suitable for all HIV infected people, and RNAi is a new attempt and a new strategy.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R512.91;R346

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