天然免疫在非典型性猴免疫缺陷病毒（SIV）感染中的作用机制探讨
发布时间：2018-11-17 07:39
【摘要】：背景在不同的个体中,人免疫缺陷病毒(HIV-1)感染导致差异性的疾病进程。95%以上的感染者出现典型性疾病进程,还有约5%HIV-1感染者出现非典型疾病进程,表现为病毒感染后,病毒载量水平维持在检测线以下或出现较晚,CD4+T细胞绝对数下降不明显,疾病进程呈现不典型性。在小剂量多次粘膜感染艾滋病猴模型研究中,研究人员也发现类似情况。部分恒河猴被猴免疫缺陷病毒(SIV)/猴-人免疫缺陷病毒(SHIV)感染后,血浆病毒载量出现明显的延迟和(或)峰值下降现象。目前研究发现细胞免疫、中和抗体等特异性免疫在非典型性SIV感染中的作用十分有限,而天然免疫一直被认为是病毒感染早期的一种重要防御机制,包括巨噬细胞的吞噬作用、NK细胞的ADCC作用以及树突细胞的抗原提呈作用等；但在非典型艾滋病毒感染过程中,天然免疫发挥作用及作用机制目尚不清楚。 此外,近来宿主体内发现一类天然免疫分子即宿主限制因子；它们是在与病毒长期共存的过程中,宿主进化出的一类抗病毒基因,包括TRIM5α、APOBEC3G、 Tetherin、SLFN11等,经体外试验均能够很好地抑制艾滋病毒。SAMHD1是最新发现的一个天然免疫分子,但恒河猴samhdl基因多态性,及对蛋白结构和功能的影响尚未见报道。 方法本研究将以SIVmac239小剂量多次直肠黏膜暴露的中国恒河猴为动物模型,通过流式细胞术等技术探究在典型和非典型的疾病进程中,CD4+T细胞、NK细胞和DC细胞的活化、增殖情况以及发挥的免疫功能,以期发现其中的免疫保护机制。另一方面,本研究通过基因测序、序列比对、蛋白质结构预测等技术,探寻恒河猴Samhd1基因上影响其蛋白功能的cSNP位点,以期更好地探索SAMHD1蛋白的抗病毒机制。 结果按照血浆病毒血症,可将SIVmac239感染恒河猴分为典型性感染、延迟性感染、隐匿性感染三组,与感染病毒量无关。隐匿组和延迟组CD4+T淋巴细胞的绝对数在整个疾病过程中要高于典型组,延迟组和典型组间有显著差异。在第14天左右三组猴都达到CD8+T淋巴细胞绝对数最低水平后,隐匿组和延迟组猴CD8+T淋巴细胞恢复的速度较快,延迟组和典型组间有显著差异。在急性感染期,典型组猴CD4+Tem比例与隐匿组猴比一直保持较高水平,具统计学差异。隐匿组CD4+Tem细胞在感染d21天后增殖活性降低且显著小于其它两组。隐匿组NK细胞的IFN-y表达量明显高于典型组,延迟组TNF-α的表达量则要明显低于其它两组猴,均有统计学差异。DC细胞释放的TNF-α水平除隐匿组在感染早期有一上调波动外,整体水平基本不变,感染39天后出现下降。对三组猴做了转录组RNA测序分析,在有极显著差异的基因中找到了与天然免疫直接相关的基因两个。 通过序列比对,在Samhd1基因上找到了11个突变位点。其中有5个位点为非同义突变,分别为15bp (C/G)、320bp (C/T)、547bp (G/A)、552bp (A/T)、839bp (T/C),确定了5个位点在SAMHD1蛋白上的位置。然后利用SNaPshot技术验证发现的这5个非同义突变位点为SNP位点,同时统计了各SNP位点的基因频率以及恒河猴Samhdl突变基因型类型和比率。最后通过生物软件预测,发现这5个非同义SNP位点影响了该蛋白的二级结构,并发现V280A位点可能影响到SAMHD1的脱氧核苷酸三磷酸水解酶催化活性。 结论非典型疾病进程的恒河猴相比典型疾病进程的恒河猴,拥有较高的CD4+T细胞绝对数水平和较快的CTL细胞恢复能力；较低的CD4+Tem细胞比例和增殖活性；较高的NK细胞通过IFN-γ发挥的免疫调节能力以及DC细胞释放的较高的TNF-α水平。这些因素发挥的抑制病毒复制作用可能是导致非典型疾病进程现象出现的重要原因。在中国恒河猴的SAMHD1蛋白中发现了至少5个SNP位点,根据预测分析发现它们的出现影响了该蛋白的功能,为更好地了解SAMHD1蛋白的抗病毒功能提供了线索。 本研究初步明确了非典型性SIV感染中天然免疫的作用及机制,对艾滋病疫苗的研发、疾病的控制具有重要的意义。
[Abstract]:Background In different individuals, human immunodeficiency virus (HIV-1) infection leads to a differential disease process. More than 95% of people living with HIV have an atypical disease process, and about 5% of the people living with HIV-1 have an atypical disease process, expressed as a viral infection, The level of viral load is below the detection line or later, the absolute number of CD4 + T cells is not obvious, and the disease process is atypical. The researchers also found similar cases in a small dose of multiple mucosal infection with the AIDS monkey model. Some of the rhesus monkeys were infected with a monkey-immune deficiency virus (SIV)/ monkey-human immunodeficiency virus (SHIV), and there was a significant delay and/ or peak reduction in plasma viral load. The present study found that specific immunity, such as cellular immunity, neutralizing antibody, and the like, is very limited in atypical SIV infection, and natural immunity has been regarded as an important defense mechanism in the early stage of viral infection, including the phagocytosis of macrophages, The role of the ADCC of NK cells as well as the antigen-extraction of dendritic cells, etc., is not clear in the process of atypical HIV infection, the role of natural immunity and the mechanism of action. in addition, a type of natural immune molecule, that is, a host restriction factor, is found in that host body, and in the process of long-term coexistence with the virus, the host has evolved a class of antiviral genes, including TRIM5, APOBEC3G, Teherin, SLFN11, and the like, The virus. SAMHD1 is the most recent discovery of a natural immune molecule, but the rhesus monkey samhdl gene polymorphism and the effect on the structure and function of the protein have not yet been met. Methods This study will study the activation and proliferation of CD4 + T cells, NK cells and DC cells in a typical and atypical disease process by flow cytometry and so on. immune function with a view to finding none of them On the other hand, by means of gene sequencing, sequence alignment, protein structure prediction and so on, the cSNP site of the rhesus monkey Samhd1 gene was found to influence its protein function, with a view to better exploring the SAMHD1 protein. Results According to the plasma viremia, the SIVmac239-infected rhesus monkey can be divided into three groups: the typical infection, the delayed infection and the occult infection. The absolute number of CD4 + T lymphocytes in the hidden group and the delayed group is higher than that of the typical group, the delay group and the code in the whole disease process, regardless of the amount of the infection virus. There was a significant difference between the groups. At the 14th day, the three groups reached the lowest level of the absolute number of CD8 + T lymphocytes, and the rate of recovery of CD8 + T lymphocytes in the hidden group and the delayed group was faster, the delay group and the dictionary There was a significant difference between the groups. In the acute stage, the proportion of CD4 + Tem in the typical group and the monkey in the hidden group remained high. The proliferation activity of the CD4 + Tem cells in the hidden group decreased after d21 infection. The expression of IFN-y in the NK cells of the group was significantly higher than that of the other two groups. The expression of TNF-y in the delayed group was significantly lower than that of the other two groups. There was a statistical difference. The level of TNF-1 released by DC cells, in addition to an up-regulated fluctuation in the early stage of the infection, was substantially the same and the infection rate was substantially unchanged. After 39 days, a decrease was observed. The three groups of monkeys were subjected to a transcriptome RNA sequencing analysis, which was found to be in a highly significant difference with the natural immunity by sequence alignment, in the Samhd1 gene, 11 mutation sites were found. Five sites were non-synonymous, 15bp (C/ G), 320bp (C/ T), 547bp (G/ A), 552bp (A/ T), and 839bp (T/ C), respectively. The position of the AMHD1 protein was determined. The five non-synonymous mutation sites identified by the SNaPshot technique were then used as the SNP site, and the gene frequency of each SNP site and the rhesus monkey Samhd were also counted. The type and ratio of the mutant genotypes were identified. Finally, the two-level structure of the protein was affected by the five non-synonymous SNP sites by the biological software prediction, and it was found that the V280A site may have an impact on the nucleus of the SAMHD1. Acid triphosphate hydrolase activity. Conclusion The rhesus monkey of the typical disease process has higher absolute number of CD4 + T cells and faster CTL cell recovery ability compared with the rhesus monkey of the typical disease process; the lower CD 4 + Tem cell ratio and proliferation activity; higher NK cells through IFN-to-play immunomodulatory capacity and DC fine The higher TNF-level of the release of the cells. These factors play a role in inhibiting the replication of the virus, which may result in the SARS At least 5 SNP sites were found in the SAMHD1 protein of the rhesus monkey in China. According to the predictive analysis, the function of the protein was affected and the SAMHD was better understood. In this study, the effect and mechanism of the natural immunity in the non-typical SIV infection were identified.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
[Abstract]:Background In different individuals, human immunodeficiency virus (HIV-1) infection leads to a differential disease process. More than 95% of people living with HIV have an atypical disease process, and about 5% of the people living with HIV-1 have an atypical disease process, expressed as a viral infection, The level of viral load is below the detection line or later, the absolute number of CD4 + T cells is not obvious, and the disease process is atypical. The researchers also found similar cases in a small dose of multiple mucosal infection with the AIDS monkey model. Some of the rhesus monkeys were infected with a monkey-immune deficiency virus (SIV)/ monkey-human immunodeficiency virus (SHIV), and there was a significant delay and/ or peak reduction in plasma viral load. The present study found that specific immunity, such as cellular immunity, neutralizing antibody, and the like, is very limited in atypical SIV infection, and natural immunity has been regarded as an important defense mechanism in the early stage of viral infection, including the phagocytosis of macrophages, The role of the ADCC of NK cells as well as the antigen-extraction of dendritic cells, etc., is not clear in the process of atypical HIV infection, the role of natural immunity and the mechanism of action. in addition, a type of natural immune molecule, that is, a host restriction factor, is found in that host body, and in the process of long-term coexistence with the virus, the host has evolved a class of antiviral genes, including TRIM5, APOBEC3G, Teherin, SLFN11, and the like, The virus. SAMHD1 is the most recent discovery of a natural immune molecule, but the rhesus monkey samhdl gene polymorphism and the effect on the structure and function of the protein have not yet been met. Methods This study will study the activation and proliferation of CD4 + T cells, NK cells and DC cells in a typical and atypical disease process by flow cytometry and so on. immune function with a view to finding none of them On the other hand, by means of gene sequencing, sequence alignment, protein structure prediction and so on, the cSNP site of the rhesus monkey Samhd1 gene was found to influence its protein function, with a view to better exploring the SAMHD1 protein. Results According to the plasma viremia, the SIVmac239-infected rhesus monkey can be divided into three groups: the typical infection, the delayed infection and the occult infection. The absolute number of CD4 + T lymphocytes in the hidden group and the delayed group is higher than that of the typical group, the delay group and the code in the whole disease process, regardless of the amount of the infection virus. There was a significant difference between the groups. At the 14th day, the three groups reached the lowest level of the absolute number of CD8 + T lymphocytes, and the rate of recovery of CD8 + T lymphocytes in the hidden group and the delayed group was faster, the delay group and the dictionary There was a significant difference between the groups. In the acute stage, the proportion of CD4 + Tem in the typical group and the monkey in the hidden group remained high. The proliferation activity of the CD4 + Tem cells in the hidden group decreased after d21 infection. The expression of IFN-y in the NK cells of the group was significantly higher than that of the other two groups. The expression of TNF-y in the delayed group was significantly lower than that of the other two groups. There was a statistical difference. The level of TNF-1 released by DC cells, in addition to an up-regulated fluctuation in the early stage of the infection, was substantially the same and the infection rate was substantially unchanged. After 39 days, a decrease was observed. The three groups of monkeys were subjected to a transcriptome RNA sequencing analysis, which was found to be in a highly significant difference with the natural immunity by sequence alignment, in the Samhd1 gene, 11 mutation sites were found. Five sites were non-synonymous, 15bp (C/ G), 320bp (C/ T), 547bp (G/ A), 552bp (A/ T), and 839bp (T/ C), respectively. The position of the AMHD1 protein was determined. The five non-synonymous mutation sites identified by the SNaPshot technique were then used as the SNP site, and the gene frequency of each SNP site and the rhesus monkey Samhd were also counted. The type and ratio of the mutant genotypes were identified. Finally, the two-level structure of the protein was affected by the five non-synonymous SNP sites by the biological software prediction, and it was found that the V280A site may have an impact on the nucleus of the SAMHD1. Acid triphosphate hydrolase activity. Conclusion The rhesus monkey of the typical disease process has higher absolute number of CD4 + T cells and faster CTL cell recovery ability compared with the rhesus monkey of the typical disease process; the lower CD 4 + Tem cell ratio and proliferation activity; higher NK cells through IFN-to-play immunomodulatory capacity and DC fine The higher TNF-level of the release of the cells. These factors play a role in inhibiting the replication of the virus, which may result in the SARS At least 5 SNP sites were found in the SAMHD1 protein of the rhesus monkey in China. According to the predictive analysis, the function of the protein was affected and the SAMHD was better understood. In this study, the effect and mechanism of the natural immunity in the non-typical SIV infection were identified.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
【参考文献】
相关期刊论文 前2条
1 李佩璐;陈倩倩;张驰宇;;髓系单核细胞来源的HIV-1限制性因子——SAMHD1[J];动物学研究;2012年05期
2 刘克剑;丛U，
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