SIVmac251不同途径感染中国恒河猴的实验研究

发布时间：2018-04-15 17:32

本文选题：SIVmac251 + 恒河猴模型　；参考：《中国协和医科大学》2007年硕士论文

【摘要】： 性传播(黏膜途径)是艾滋病的主要传播途径之一，建立粘膜感染动物模型具有非常重要的意义。目前，国内缺乏对静脉和粘膜感染的对比性研究，本实验用SIVmac251经静脉、阴道和直肠三种途径感染恒河猴，对SIV的感染情况、不同感染途径是否对临床表现、病毒学指标及基因变异上存在影响以及影响程度等问题进行了实验研究。 SIVmac251通过静脉、阴道和直肠三种途径各感染恒河猴1只，通过对病毒分离、血浆病毒载量、PBMC中前病毒DNA检测、CD4~+／CD8~+比值等一系列指标的测定，表明三种途径均成功感染实验猴，同时建立了黏膜感染动物模型。临床发现实验猴在感染后14天出现食欲减退，并持续两周左右，体重有所下降，腹股沟等处浅表淋巴结肿大。静脉感染猴S153感染后半年时，食欲减退，体重下降，腹泻，腹股沟淋巴结变小，六个半月时死亡。用SYBR Green I荧光染料实时定量RT-PCR方法检测病毒，各途径感染病毒血症出现时间有先后差别，分别为：静脉3d、直肠7d和阴道10d，没有观察到高峰期延迟现象，都是在14d达到高峰，以后迅速下降。用病毒分离的方法，两只粘膜感染猴S142和S172较静脉感染猴S153有高峰期延长现象。抗体检测结果显示，静脉感染猴在感染后10d时抗体转为阳性，整个感染过程中抗体水平都很低，最高滴度为1：80；两只黏膜感染猴在14d时抗体转为阳性，整个实验期抗体水平一直比较高，阴道感染猴S142最高到1：5120，直肠感染猴S172最高到1：2560。同时发现，在42w时两只黏膜感染猴组织中病毒载量很高，阴道感染猴1.118E+08，直肠感染猴1.288E+07；淋巴结病理切片显示，处于病程进展期；这时血浆中病毒载量却很低，阴道感染猴4.322E+03，直肠感染猴为阴性。 CD4~+／CD8~+比值和CD4~+细胞数变化趋势基本一致，在28d时CD4~+／CD8~+比值倒置。 nef和env v1v2两段基因在病毒感染急性期中基因变异研究，从测序结果看，三种途径感染后在env段没有表现出很大的差别；血浆中nefRNA和DNA的变异情况都表现出了不同途径上的差异，三种途径nefRNA均在两个位点发生稳定的突变，静脉感染猴在nefDNA中却没有稳定突变的产生。阴道感染猴S142和直肠感染猴S172 nef DNA和RNA分别在很多位点都发生了突变，这些位点并不相同。 SIV不同途径成功感染中国恒河猴和SIV不同途径感染后的实验研究，对于更好的认识艾滋病，解读发病机制，对于疫苗设计及评价，都具有非常重要的理论及现实意义。由于条件有限，本实验共感染恒河猴3只，每种途径1只，，所得实验数据不能排除个体差异和一些别的因素所带来的影响，结果仅对不同途径感染进行初步探索性研究，有待于进一步更为详尽的研究。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332

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