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  本文選題：SIVmac251 + 恒河猴模型��； 參考：《中國協(xié)和醫(yī)科大學》2007年碩士論文

【摘要】： 性傳播(黏膜途徑)是艾滋病的主要傳播途徑之一，建立粘膜感染動物模型具有非常重要的意義。目前，國內(nèi)缺乏對靜脈和粘膜感染的對比性研究，本實驗用SIVmac251經(jīng)靜脈、陰道和直腸三種途徑感染恒河猴，對SIV的感染情況、不同感染途徑是否對臨床表現(xiàn)、病毒學指標及基因變異上存在影響以及影響程度等問題進行了實驗研究。 SIVmac251通過靜脈、陰道和直腸三種途徑各感染恒河猴1只，通過對病毒分離、血漿病毒載量、PBMC中前病毒DNA檢測、CD4~+／CD8~+比值等一系列指標的測定，表明三種途徑均成功感染實驗猴，同時建立了黏膜感染動物模型。 臨床發(fā)現(xiàn)實驗猴在感染后14天出現(xiàn)食欲減退，并持續(xù)兩周左右，體重有所下降，腹股溝等處淺表淋巴結(jié)腫大。靜脈感染猴S153感染后半年時，食欲減退，體重下降，腹瀉，腹股溝淋巴結(jié)變小，六個半月時死亡。用SYBR Green I熒光染料實時定量RT-PCR方法檢測病毒，各途徑感染病毒血癥出現(xiàn)時間有先后差別，分別為：靜脈3d、直腸7d和陰道10d，沒有觀察到高峰期延遲現(xiàn)象，都是在14d達到高峰，以后迅速下降。用病毒分離的方法，兩只粘膜感染猴S142和S172較靜脈感染猴S153有高峰期延長現(xiàn)象。 抗體檢測結(jié)果顯示，靜脈感染猴在感染后10d時抗體轉(zhuǎn)為陽性，整個感染過程中抗體水平都很低，最高滴度為1：80；兩只黏膜感染猴在14d時抗體轉(zhuǎn)為陽性，整個實驗期抗體水平一直比較高，陰道感染猴S142最高到1：5120，直腸感染猴S172最高到1：2560。 同時發(fā)現(xiàn)，在42w時兩只黏膜感染猴組織中病毒載量很高，陰道感染猴1.118E+08，直腸感染猴1.288E+07；淋巴結(jié)病理切片顯示，處于病程進展期；這時血漿中病毒載量卻很低，陰道感染猴4.322E+03，直腸感染猴為陰性。 CD4~+／CD8~+比值和CD4~+細胞數(shù)變化趨勢基本一致，在28d時CD4~+／CD8~+比值倒置。 nef和env v1v2兩段基因在病毒感染急性期中基因變異研究，從測序結(jié)果看，三種途徑感染后在env段沒有表現(xiàn)出很大的差別；血漿中nefRNA和DNA的變異情況都表現(xiàn)出了不同途徑上的差異，三種途徑nefRNA均在兩個位點發(fā)生穩(wěn)定的突變，靜脈感染猴在nefDNA中卻沒有穩(wěn)定突變的產(chǎn)生。陰道感染猴S142和直腸感染猴S172 nef DNA和RNA分別在很多位點都發(fā)生了突變，這些位點并不相同。 SIV不同途徑成功感染中國恒河猴和SIV不同途徑感染后的實驗研究，對于更好的認識艾滋病，解讀發(fā)病機制，對于疫苗設計及評價，都具有非常重要的理論及現(xiàn)實意義。由于條件有限，本實驗共感染恒河猴3只，每種途徑1只，，所得實驗數(shù)據(jù)不能排除個體差異和一些別的因素所帶來的影響，結(jié)果僅對不同途徑感染進行初步探索性研究，有待于進一步更為詳盡的研究。
[Abstract]:Sexual transmission (mucosal pathway) is one of the main transmission routes of AIDS. It is of great significance to establish animal models of mucosal infection.At present, there is a lack of comparative study on venous and mucosal infection in China. In this study, we used SIVmac251 to infect rhesus monkey via vein, vagina and rectum.The influence of virology index and gene variation was studied experimentally.One rhesus monkey was infected by SIVmac251 via vein, vagina and rectum. A series of indexes such as CD _ 4 ~ / CD _ 8 ~ ratio were detected by DNA in plasma virus load and the results showed that all the three ways were successful in infecting experimental monkey.At the same time, the animal model of mucosal infection was established.It was found that the experimental monkey had anorexia 14 days after infection, which lasted for about two weeks, with weight loss and superficial lymphadenopathy in groin and so on.Six months after S153 infection, the monkeys suffered from loss of appetite, weight loss, diarrhea, small inguinal lymph nodes, and died six and a half months later.SYBR Green I fluorescence dye real-time quantitative RT-PCR method was used to detect the virus. The time of viremia infection was different in different ways, which were 3 days of vein, 7 days of rectum and 10 days of vagina. No peak delay was observed, and the peak time was reached at 14 days.After a rapid decline.By the method of virus isolation, the peak periods of S142 and S172 in two infected monkeys were longer than those in veno-infected monkeys.The results of antibody test showed that the antibody turned positive at 10 days after infection, and the antibody level was very low in the whole infection process, the highest titer was 1: 80, and the antibody became positive at 14 days in two monkeys infected with mucous membrane.The antibody level was high throughout the experimental period, with the highest S142 to 1: 5120 for vaginal infection and the highest to 1: 2560 for rectal infection.It was also found that the viral load in the tissues of the two monkeys infected with mucous membrane was very high at 42w, 1.118E08in vagina and 1.288E07in rectum. The pathological sections of lymph nodes showed that they were in the advanced stage of disease, but the viral load in plasma was very low.The vaginal infection monkey was 4.322 E 03, and the rectal infection monkey was negative.The ratio of CD4 ~ / / CD8 ~ ~ and the number of CD4 ~ ~ cells were basically the same, and the ratio of CD4 ~ / / CD8 ~ ~ was inverted at 28 days.The genetic variation of nef and env v1v2 genes in the acute stage of viral infection was studied. The results of sequencing showed that there was no significant difference in the env segment between the three pathways after infection, and the variation of nefRNA and DNA in plasma showed differences in different pathways.Stable mutations of nefRNA were found at two loci in all three pathways, but no stable mutations were found in veno-infected monkeys in nefDNA.S142 and S172 nef DNA and RNA have mutated at many sites, which are different.The experimental study on the successful infection of Chinese rhesus monkey and SIV in different ways of SIV is of great theoretical and practical significance for better understanding of AIDS, understanding the pathogenesis of AIDS, and for vaccine design and evaluation.Due to limited conditions, three rhesus monkeys were infected with one rhesus monkey per pathway. The experimental data could not exclude the influence of individual differences and some other factors.Further and more detailed studies are needed.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R-332

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本文編號：1755096