本文選題：人類免疫缺陷病毒(HIV) + 艾滋病　； 參考：《復旦大學》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,艾滋病的流行已經給國家經濟和社會帶來了巨大負擔。但目前的抗病毒藥物無法給患者提供徹底的治療,因此更多的研究開始轉變以往的治療思路,探索新的更有效更經濟的艾滋病治療方法。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干擾效應是指雙鏈RNA在生物細胞內對序列特異性基因轉錄和表達的抑制作用。RNAi的研究為控制HIV感染帶來希望,通過SiRNA對病毒生活周期的多個水平作用,可顯著抑制病毒的復制。目前已有研究報道了通過對HIV-1 gag,tat,rev和宿主CD4及CCR5受體進行RNA干擾,進而達到抑制HIV病毒復制的目的,并且研究取得了一定的進展,為抗HIV治療開辟了一條新路。 HIV-1的特點之一是具有多個調節(jié)基因,這些基因可編碼相應的調控蛋白,如Nef、Tat、Rev、Vif、Vpr、Vpx、Vpu等,它們在RNA轉錄、轉錄后加工、蛋白質翻譯直到病毒顆粒從細胞膜釋放的過程中發(fā)揮重要的調節(jié)作用。Vif蛋白被歸于附屬蛋白質,編碼一個含有192個氨基酸,相對分子質量大約23000的胞質蛋白,vif基因比HIV-1基因組中env、gag等突變率高的基因相對保守。但10多年前就已經知道Vif是HIV-1感染所必須的調控蛋白,vif基因缺陷性(△vif)的HIV-1和SIV在體內無法復制,在體外雖可正常復制并產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)學上也表現(xiàn)異常。在子彈頭狀病毒核心的電子密集物質明顯減少,而核心與包膜之間的空間里電子密集物質明顯增加,使核心形狀呈多樣化。另外,在感染細胞中的反轉錄過程以及病毒顆粒中的內源性反轉錄也是明顯受損,因而不能感染靶細胞。有趣的是,vif缺陷性的病毒在一些腫瘤細胞系可以順利的進行復制。根據△vif HIV-1是否能夠在其內復制可將細胞分為“允許細胞”和“非允許細胞”�！鱲if HIV-1病毒可以進入非允許細胞中,但在反轉錄過程中或反轉錄之后到整合之前由于某種尚不清楚的機制導致感染終止。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結構及蛋白表達等方面均未發(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APOBEC3G與Vif相互作用機制的深入研究,使越來越多的研究者開始重視vif基因及其表達產物在HIV生命周期中的作用。 經過科研人員數(shù)年的努力,現(xiàn)在RNA干擾的機制已經得到比較清楚的闡述。RNAi是一種進化上保守的抵御轉基因或外來病毒侵犯的防御機制,是指內源性或外源性大約為21～23bp的核苷酸與靶基因的轉錄產物mRNA存在同源互補序列的雙鏈RNA(double-stranded RNA,dsRNA)在細胞內特異地降解該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錄后基因沉默(post-transcriptional gene silencing,PTGS)。RNAi作為一種簡單、快速、特異、高效、經濟、效果可預測的技術,具有明顯的優(yōu)點,它比反義核酸技術優(yōu)越,比基因敲除簡單,具有很好的應用前景。具體可用于基因功能分析、研究信號傳導通路的新途徑、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互相產生干擾,效果更好,相信隨著對RNAi機制的進一步認識,這一技術能夠成為抗病毒,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,通過設計針對vif的特異性SiRNA對HIV病毒RNA進行干擾降解,進而達到降低或抑制病毒復制的目的。研究中我們成功的設計了3段vif特異性SiRNA,對轉接入pEGFP-N1質粒中的vif基因進行干擾,并分別在核酸和蛋白水平進行了驗證,結果證明了RNAi可以有效且特異性的下調Vif蛋白的表達。 本課題的研究目的就是利用RNAi技術對HIV-1 vif基因的轉錄水平進行下調,達到抑制Vif蛋白表達的目的,為新的抗HIV治療和預防研究提供理論基礎。研究首先選擇人胚腎293T(HEK 293T)細胞作為宿主細胞,EGFP融合蛋白作為報告基因,觀察HIV-1 Vif蛋白在細胞內的表達情況,為后續(xù)的RNAi做好準備。第一部分先建立pEGFP-N1-vif融合質粒,進行HEK 293T細胞轉染,熒光顯微鏡觀察轉染細胞中的轉染效率,蛋白印跡免疫分析技術(Western blot)檢測Vif蛋白的表達情況。結果顯示,HIV-1 Vif可以在HEK 293T細胞中成功表達。采用體外轉錄法合成SiRNA,首先合成六段SiRNA(四段針對vif和一段陽性對照、一段陰性對照)的正反義鏈模板,體外進行反轉錄,得到長21bp的雙鏈SiRNA,然后雙鏈SiRNA分別與pEGFP-N1-vif融合質粒共轉染HEK 293T細胞。熒光下觀察綠色熒光,篩選出能抑制HIV-1 vif基因表達的有效SiRNA。驗證轉染成功后進行細胞抽提總RNA,進行實時定量RT-PCR(Real-time PCR),驗證干擾效果,Western blot檢測蛋白表達情況。結果顯示,與陰性對照比較,有3條SiRNA可明顯抑制vifmRNA的表達,S-SiRNA1、S-SiRNA2組和S-SiRNA3分別下降了約53%、60%和63%;同時結果顯示蛋白表達水平也明顯降低,3條SiRNA的蛋白條帶均明顯減弱。最終驗證了RNAi可以對HIV-1 vif進行有效且特異性的下調表達。 本實驗成功的建立了哺乳動物細胞pEGFP-N1-HIV-1/vif表達系統(tǒng),并得以驗證,Vif蛋白可以在體外哺乳動物細胞中高水平表達。研究應用RNAi技術,對HIV-1 vif從轉錄和表達水平進行了沉默,并分別在核酸和蛋白水平進行了驗證,這對于阻斷病毒和宿主細胞結合及相互作用有重要啟示,表明vif可以作為進一步研究抗HIV-1的潛力靶點,也為這一基因水平治療手段的開展進行了積累。同時證實,RNAi作為一種簡單、快速、特異、高效、經濟、效果可預測的技術,具有明顯的優(yōu)點。但值得注意的是,本實驗中的SiRNA也不能實現(xiàn)對HIV-1 vif的徹底敲除,干擾效率維持在50%左右。曾有研究報道,RNAi對HIV-1的干擾效率可以達到80%～95%左右。本實驗考慮可能與實驗操作和技術有一定的關系,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技術的問世是生命科學發(fā)展史上的一大豐碑,若能長期穩(wěn)定應用于抗病毒治療,尤其是抗HIV治療,無疑將造福人類。雖然目前RNAi技術在抗病毒的實驗研究方面已取得可喜成績,但由于諸多問題的存在,其應用于臨床還有一段艱難漫長的道路。從理論和技術兩個層面來解決RNAi的分子基礎及其治療應用問題任重而道遠,因此對此項技術還需進一步的改進和創(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—反轉錄酶和蛋白酶—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間后,體內病毒產生了耐藥性。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。有研究者預期,新型療法或許可以與現(xiàn)行的反轉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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R512.91;R346

【相似文獻】

相關期刊論文 前10條

1 李月敏,姜明紅,錢其軍,宋三泰;RNAi技術在病毒性疾病和腫瘤治療中的應用前景[J];軍事醫(yī)學科學院院刊;2004年03期

2 郭驍才,郭紅霞;冠狀病毒的siRNA設計[J];應用與環(huán)境生物學報;2003年05期

3 賀娟;呂回;;小分子干擾RNA在細胞凋亡調控中的應用研究進展[J];醫(yī)學綜述;2007年03期

4 劉海防,謝青;RNA干擾抗艾滋病病毒感染研究進展[J];國外醫(yī)學.流行病學.傳染病學分冊;2005年03期

5 劉莉,謝曉華,常連慶;應用siRNA抑制由醛固酮誘導的大鼠肥大心肌細胞中鈣調神經磷酸酶AβmRNA的表達[J];心臟雜志;2005年04期

6 李陽;倪兵;;RNA干擾與人類疾病[J];第三軍醫(yī)大學學報;2005年23期

7 金存嬌;;基因水平上的抗病毒新技術——RNAi[J];亞太傳統(tǒng)醫(yī)藥;2009年08期

8 鄧娟,楊益良,付士紅,王力華,金冬雁,梁國棟;RNAi抑制XJ-160病毒復制的研究[J];中華微生物學和免疫學雜志;2005年06期

9 戴嵐;狄文;;小干擾RNA在卵巢腫瘤治療中的應用前景[J];國外醫(yī)學(婦產科學分冊);2006年05期

10 王井偉;曹磊;付軍;田海梅;劉義;張偉;;RNAi對骨肉瘤MG-63細胞survivin基因表達抑制作用[J];中國癌癥雜志;2007年02期

相關會議論文 前10條

1 楊群輝;范志斌;胡建民;楊建成;;siRNA介導的RNAi對心肌細胞�；撬徂D運體基因抑制效果的研究[A];全國動物生理生化第十一次學術交流會論文摘要匯編[C];2010年

2 楊群輝;楊碩;胡建民;楊建成;;siRNA介導的RNAi對心肌細胞半胱亞磺酸脫羧酶基因抑制效果的研究[A];全國動物生理生化第十一次學術交流會論文摘要匯編[C];2010年

3 程娜;;RNA干擾與基因治療[A];中國生物醫(yī)學工程進展——2007中國生物醫(yī)學工程聯(lián)合學術年會論文集（下冊）[C];2007年

4 鄧清華;劉國文;劉磊;王建國;朱曉巖;李小兵;王哲;;siRNA特異性抑制犢牛原代肝細胞SREBP-1c基因的表達[A];中國畜牧獸醫(yī)學會家畜內科學分會第七屆代表大會暨學術研討會論文集（上冊）[C];2011年

5 馬蕾娜;錢程;;單一polⅡ啟動子調控抗環(huán)氧化酶-siRNA和TRAIL蛋白對肝癌的協(xié)同治療[A];中華醫(yī)學會病理學分會2010年學術年會日程及論文匯編[C];2010年

6 季愛民;蘇丹;車鷗;李文適;孫靚;張忠義;楊彬;;殼聚糖/siRNA納米粒介導的功能性基因沉默研究(英文)[A];2010年中國藥學大會暨第十屆中國藥師周論文集[C];2010年

7 黃偉;呂明;金明姬;楊長青;高鐘鎬;;mPEG-PCL-g-PEI聚合物的合成及其siRNA遞送性能[A];2010年中國藥學大會暨第十屆中國藥師周論文集[C];2010年

8 陳宇;鄭海濤;楊力建;陳少華;沈琦;劉思初;李，

本文編號：2012793