天然免疫在非典型性猴免疫缺陷病毒（SIV）感染中的作用機(jī)制探討
發(fā)布時(shí)間：2018-11-17 07:39
【摘要】：背景在不同的個(gè)體中,人免疫缺陷病毒(HIV-1)感染導(dǎo)致差異性的疾病進(jìn)程。95%以上的感染者出現(xiàn)典型性疾病進(jìn)程,還有約5%HIV-1感染者出現(xiàn)非典型疾病進(jìn)程,表現(xiàn)為病毒感染后,病毒載量水平維持在檢測(cè)線以下或出現(xiàn)較晚,CD4+T細(xì)胞絕對(duì)數(shù)下降不明顯,疾病進(jìn)程呈現(xiàn)不典型性。在小劑量多次粘膜感染艾滋病猴模型研究中,研究人員也發(fā)現(xiàn)類(lèi)似情況。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,血漿病毒載量出現(xiàn)明顯的延遲和(或)峰值下降現(xiàn)象。目前研究發(fā)現(xiàn)細(xì)胞免疫、中和抗體等特異性免疫在非典型性SIV感染中的作用十分有限,而天然免疫一直被認(rèn)為是病毒感染早期的一種重要防御機(jī)制,包括巨噬細(xì)胞的吞噬作用、NK細(xì)胞的ADCC作用以及樹(shù)突細(xì)胞的抗原提呈作用等；但在非典型艾滋病毒感染過(guò)程中,天然免疫發(fā)揮作用及作用機(jī)制目尚不清楚。 此外,近來(lái)宿主體內(nèi)發(fā)現(xiàn)一類(lèi)天然免疫分子即宿主限制因子；它們是在與病毒長(zhǎng)期共存的過(guò)程中,宿主進(jìn)化出的一類(lèi)抗病毒基因,包括TRIM5α、APOBEC3G、 Tetherin、SLFN11等,經(jīng)體外試驗(yàn)均能夠很好地抑制艾滋病毒。SAMHD1是最新發(fā)現(xiàn)的一個(gè)天然免疫分子,但恒河猴samhdl基因多態(tài)性,及對(duì)蛋白結(jié)構(gòu)和功能的影響尚未見(jiàn)報(bào)道。 方法本研究將以SIVmac239小劑量多次直腸黏膜暴露的中國(guó)恒河猴為動(dòng)物模型,通過(guò)流式細(xì)胞術(shù)等技術(shù)探究在典型和非典型的疾病進(jìn)程中,CD4+T細(xì)胞、NK細(xì)胞和DC細(xì)胞的活化、增殖情況以及發(fā)揮的免疫功能,以期發(fā)現(xiàn)其中的免疫保護(hù)機(jī)制。另一方面,本研究通過(guò)基因測(cè)序、序列比對(duì)、蛋白質(zhì)結(jié)構(gòu)預(yù)測(cè)等技術(shù),探尋恒河猴Samhd1基因上影響其蛋白功能的cSNP位點(diǎn),以期更好地探索SAMHD1蛋白的抗病毒機(jī)制。 結(jié)果按照血漿病毒血癥,可將SIVmac239感染恒河猴分為典型性感染、延遲性感染、隱匿性感染三組,與感染病毒量無(wú)關(guān)。隱匿組和延遲組CD4+T淋巴細(xì)胞的絕對(duì)數(shù)在整個(gè)疾病過(guò)程中要高于典型組,延遲組和典型組間有顯著差異。在第14天左右三組猴都達(dá)到CD8+T淋巴細(xì)胞絕對(duì)數(shù)最低水平后,隱匿組和延遲組猴CD8+T淋巴細(xì)胞恢復(fù)的速度較快,延遲組和典型組間有顯著差異。在急性感染期,典型組猴CD4+Tem比例與隱匿組猴比一直保持較高水平,具統(tǒng)計(jì)學(xué)差異。隱匿組CD4+Tem細(xì)胞在感染d21天后增殖活性降低且顯著小于其它兩組。隱匿組NK細(xì)胞的IFN-y表達(dá)量明顯高于典型組,延遲組TNF-α的表達(dá)量則要明顯低于其它兩組猴,均有統(tǒng)計(jì)學(xué)差異。DC細(xì)胞釋放的TNF-α水平除隱匿組在感染早期有一上調(diào)波動(dòng)外,整體水平基本不變,感染39天后出現(xiàn)下降。對(duì)三組猴做了轉(zhuǎn)錄組RNA測(cè)序分析,在有極顯著差異的基因中找到了與天然免疫直接相關(guān)的基因兩個(gè)。 通過(guò)序列比對(duì),在Samhd1基因上找到了11個(gè)突變位點(diǎn)。其中有5個(gè)位點(diǎn)為非同義突變,分別為15bp (C/G)、320bp (C/T)、547bp (G/A)、552bp (A/T)、839bp (T/C),確定了5個(gè)位點(diǎn)在SAMHD1蛋白上的位置。然后利用SNaPshot技術(shù)驗(yàn)證發(fā)現(xiàn)的這5個(gè)非同義突變位點(diǎn)為SNP位點(diǎn),同時(shí)統(tǒng)計(jì)了各SNP位點(diǎn)的基因頻率以及恒河猴Samhdl突變基因型類(lèi)型和比率。最后通過(guò)生物軟件預(yù)測(cè),發(fā)現(xiàn)這5個(gè)非同義SNP位點(diǎn)影響了該蛋白的二級(jí)結(jié)構(gòu),并發(fā)現(xiàn)V280A位點(diǎn)可能影響到SAMHD1的脫氧核苷酸三磷酸水解酶催化活性。 結(jié)論非典型疾病進(jìn)程的恒河猴相比典型疾病進(jìn)程的恒河猴,擁有較高的CD4+T細(xì)胞絕對(duì)數(shù)水平和較快的CTL細(xì)胞恢復(fù)能力；較低的CD4+Tem細(xì)胞比例和增殖活性；較高的NK細(xì)胞通過(guò)IFN-γ發(fā)揮的免疫調(diào)節(jié)能力以及DC細(xì)胞釋放的較高的TNF-α水平。這些因素發(fā)揮的抑制病毒復(fù)制作用可能是導(dǎo)致非典型疾病進(jìn)程現(xiàn)象出現(xiàn)的重要原因。在中國(guó)恒河猴的SAMHD1蛋白中發(fā)現(xiàn)了至少5個(gè)SNP位點(diǎn),根據(jù)預(yù)測(cè)分析發(fā)現(xiàn)它們的出現(xiàn)影響了該蛋白的功能,為更好地了解SAMHD1蛋白的抗病毒功能提供了線索。 本研究初步明確了非典型性SIV感染中天然免疫的作用及機(jī)制,對(duì)艾滋病疫苗的研發(fā)、疾病的控制具有重要的意義。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.91
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 李佩璐;陳倩倩;張馳宇;;髓系單核細(xì)胞來(lái)源的HIV-1限制性因子——SAMHD1[J];動(dòng)物學(xué)研究;2012年05期
2 劉克劍;叢U，
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