HIV感染中Th17細(xì)胞與消化道上皮屏障完整性關(guān)系的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-07-13 17:49
【摘要】：CD4~+T細(xì)胞缺失和機(jī)體持續(xù)的免疫激活是HIV感染的主要免疫病理特征。病毒感染導(dǎo)致腸道免疫系統(tǒng)結(jié)構(gòu)和功能的損傷、粘膜屏障破壞、微生物易位,從而導(dǎo)致機(jī)體慢性免疫活化,這可能是HIV-1致病的重要機(jī)制。雖然HIV感染過(guò)程中Th17細(xì)胞發(fā)生顯著變化,但該細(xì)胞的缺失對(duì)免疫系統(tǒng)的影響尚不清楚,尤其是Th17細(xì)胞在粘膜屏障損傷中的作用亟待研究。本文應(yīng)用SIV/SHIV感染恒河猴分析消化道粘膜CD4~+T Th17細(xì)胞與消化道粘膜屏障結(jié)構(gòu)在病毒感染后的變化及相互關(guān)系;同時(shí)應(yīng)用Caco-2細(xì)胞體外構(gòu)建細(xì)胞單層,探討SIV/SHIV蛋白組分gp160和Tat以及IL-17對(duì)上皮細(xì)胞屏障的影響,為HIV-1感染所致粘膜屏障損傷的修復(fù)提供新思路。在體內(nèi)實(shí)驗(yàn)研究中,為了解SIV/SHIV感染對(duì)恒河猴消化道粘膜CD4~+TTh17細(xì)胞的影響,本文采用流式細(xì)胞術(shù)檢測(cè)了 CD3+CD4~+T細(xì)胞、IL-17+CD3+CD4~+T細(xì)胞、IL-10+CD3+CD4~+T 細(xì)胞、Foxp3+CD3+CD4~+T 細(xì)胞、IL-4+CD3+CD4~+T 細(xì)胞和IFNy+CD3+CD4~+T細(xì)胞亞群的相對(duì)大小;并通過(guò)realtimeRT-PCR檢測(cè)了IL-17A、RORyt、TGF-β、GATA-3、T-bet和Foxp3等基因mRNA表達(dá)水平在病毒感染后的變化。為了解粘膜屏障在SIV/SHIV感染后的變化,本文利用透射電子顯微鏡觀察了正常動(dòng)物和感染動(dòng)物消化道粘膜上皮的超微結(jié)構(gòu),采用real time RT-PCR檢測(cè)了與緊密連接相關(guān)的claudin-1,claudin-3,occludin和ZO-1基因的轉(zhuǎn)錄水平,應(yīng)用western blot和免疫熒光染色技術(shù)檢測(cè)了緊密連接相關(guān)的相關(guān)蛋白Claudin-1,Claudin-3,Occludin和ZO-1的表達(dá)水平。為驗(yàn)證IL-17A的保護(hù)作用,本文采用嵌入式transwell培養(yǎng)Caco-2細(xì)胞,體外構(gòu)建細(xì)胞單層,觀察了 SHIV/SIV蛋白組分gp160和Tat以及IL-17對(duì)上皮細(xì)胞屏障跨上皮細(xì)胞電阻、FITC-Dextran通透性的影響,同時(shí)應(yīng)用real time RT-PCR檢測(cè)了細(xì)胞單層中與緊密連接相關(guān)的claudin-1,claudin-3和ZO-1基因的轉(zhuǎn)錄水平。研究發(fā)現(xiàn),SIV/SHIV感染動(dòng)物消化道粘膜中CD3+CD4~+T細(xì)胞比例較未感染動(dòng)物低;IFNγ+CD3+CD4~+T細(xì)胞和IL-4+CD3+CD4~+T細(xì)胞比例較未感染動(dòng)物的高,Th1細(xì)胞和Th2細(xì)胞核轉(zhuǎn)錄因子T-bet和GATA-3的轉(zhuǎn)錄水平與未感染動(dòng)物相比有不同程度的升高;在病毒感染組腸道粘膜組織中,除回腸,IL-17+CD3+CD4~+T細(xì)胞比例較正常組降低,三組感染動(dòng)物消化道粘膜中IL-17的轉(zhuǎn)錄水平均低于正常動(dòng)物;檢測(cè) IL-10+CD3+CD4~+ T 細(xì)胞和 Foxp3+CD3+CD4~+ T 細(xì)胞,SIV/SHIV感染動(dòng)物除結(jié)腸Foxp3+CD3+CD4~+T細(xì)胞比例較正常動(dòng)物降低,其余消化道粘膜組織中這兩類細(xì)胞比例較正常動(dòng)物均升高;real time RT-PCR檢測(cè)Foxp3結(jié)果顯示,除Gr4組空腸、盲腸和結(jié)腸Foxp3轉(zhuǎn)錄水平與正常動(dòng)物相比較降低,另兩組SHIV感染動(dòng)物消化道粘膜組織中Foxp3轉(zhuǎn)錄水平與正常動(dòng)物相比較高。在研究動(dòng)物消化道上皮屏障的過(guò)程中,通過(guò)透射電子顯微鏡觀察Gr4組和Gr19組動(dòng)物小腸和大腸粘膜上皮細(xì)胞超微結(jié)構(gòu),與正常動(dòng)物進(jìn)行對(duì)比,可見(jiàn)上皮細(xì)胞間緊密連接增寬,胞間縫隙增大:微絨毛排列散亂、變細(xì)、稀疏,呈現(xiàn)倒伏狀,有部分?jǐn)嗔�、脫�?內(nèi)質(zhì)網(wǎng)有擴(kuò)張現(xiàn)象,線粒體有腫脹,在胞質(zhì)內(nèi)有空泡出現(xiàn)。在基因水平,感染動(dòng)物中claudin-1的轉(zhuǎn)錄水平較正常組均降低,直腸中Gr19組claudin-1的轉(zhuǎn)錄水平較正常組降低;蛋白水平,感染組動(dòng)物的空腸、盲腸和結(jié)腸粘膜中Claudin-1的蛋白表達(dá)水平較正常組降低,激光共聚焦顯微鏡觀察發(fā)現(xiàn)病毒感染組動(dòng)物消化道粘膜組織中Claudin-1的分布和蛋白表達(dá)水平均受到影響,分布散亂、熒光度低。Claudin-3在基因水平,感染動(dòng)物十二指腸和空腸claudin-3的轉(zhuǎn)錄水平較正常組均降低;感染動(dòng)物回腸、結(jié)腸和直腸中Gr19組claudin-3的轉(zhuǎn)錄水平較正常組均降低;蛋白水平感染動(dòng)物的空腸、結(jié)腸和直腸粘膜中Claudin-3的蛋白表達(dá)水平較正常組降低;十二指腸中Gr19組Claudin-3的表達(dá)水平較正常組升高,Gr15組降低;盲腸中Gr19組和Gr15組Claudin-3的蛋白表達(dá)水平較正常組降低。Real-time RT PCR檢測(cè)發(fā)現(xiàn)在感染動(dòng)物十二指腸、空腸和直腸occludin的轉(zhuǎn)錄水平較正常組均降低;western blot檢測(cè)發(fā)現(xiàn)感染動(dòng)物小腸粘膜中Occludin的蛋白表達(dá)水平均較低,結(jié)腸中Gr19組和Gr4組Occludin的蛋白表達(dá)水平較正常組降低。Real-timeRTPCR檢測(cè)發(fā)現(xiàn)在感染動(dòng)物十二指腸和空腸ZO-1的轉(zhuǎn)錄水平較正常組均降低;激光共聚焦顯微鏡觀察動(dòng)物消化道粘膜組織免疫熒光染色切片,發(fā)現(xiàn)病毒感染組動(dòng)物消化道粘膜組織中ZO-1的分布和蛋白表達(dá)水平均受到影響,分布不連續(xù)、熒光度降低。在體外實(shí)驗(yàn)研究中發(fā)現(xiàn),我們可以建立穩(wěn)定的Caco-2細(xì)胞體外研究體系,Tat(14ng/ml)對(duì)細(xì)胞單層具有即時(shí)影響效應(yīng),即作用2h后會(huì)增加細(xì)胞單層通透性并降低TER;特定濃度Tat和gp1 60對(duì)Caco-2細(xì)胞單層緊密連接相關(guān)基因表達(dá)的影響不大;IL-17(100ng/ml)對(duì)細(xì)胞單層有一定程度的保護(hù)作用。綜上所述,SIV/SHIV感染恒河猴消化道粘膜CD3+CD4~+ T細(xì)胞和IL-17+CD3+CD4~+T 細(xì)胞比例下降,與緊密連接相關(guān)的 claudin-1,claudin-3,occludin和ZO-1基因的轉(zhuǎn)錄水平以及相關(guān)蛋白的表達(dá)水平有不同程度的下降,Tat(14ng/ml)對(duì)細(xì)胞單層具有即時(shí)影響效應(yīng),IL-17(100ng/ml)對(duì)細(xì)胞單層有一定程度的保護(hù)作用,這些結(jié)果為新的治療策略的發(fā)現(xiàn)、探索新的方法減少微生物易位和免疫激活提供新思路。
[Abstract]:The loss of CD4~+T cells and the continuous immune activation of the body are the main immuno pathological features of HIV infection. Virus infection leads to damage of the structure and function of the intestinal immune system, the destruction of the mucosal barrier, and the translocation of microorganisms, which may lead to the chronic immune activation of the body. This may be an important mechanism for the pathogenesis of HIV-1. Although Th17 cells are infected during the HIV infection process. The effect of the loss of the cells on the immune system is not clear, especially the role of Th17 cells in the mucosal barrier damage. In this paper, the changes and relationships of the CD4~+T Th17 cells in the digestive tract mucosa and the mucosal barrier structure in the digestive tract were analyzed by SIV/SHIV infection in Ganges RIver monkey. At the same time, Ca was applied to the infection of the mucosal barrier. The cell monolayers of CO-2 cells were constructed in vitro, and the effects of SIV/SHIV protein components gp160 and Tat and IL-17 on the epithelial barrier were provided to provide new ideas for the repair of mucosal barrier damage caused by HIV-1 infection. In vivo, in order to understand the effect of SIV/SHIV infection on the mucous membrane CD4~+TTh17 cells of the digestive tract of Ganges RIver monkey, the flow finer was used in this paper. Cytoplasm was used to detect the relative size of CD3+CD4~+T, IL-17+CD3+CD4~+T, IL-10+CD3+CD4~+T, Foxp3+CD3+CD4~+T, IL-4+CD3+CD4~+T and IFNy+CD3+CD4~+T cells, and the expression levels of IL-17A, RORyt, TGF- beta, GATA-3, T-bet, and IFNy+CD3+CD4~+T were detected by realtimeRT-PCR. In order to understand the changes in the mucosal barrier after SIV/SHIV infection, the ultrastructure of the mucosal epithelium in the digestive tract of normal and infected animals was observed by transmission electron microscopy. The transcriptional level of the claudin-1, Claudin-3, occludin and ZO-1 genes related to the close connection was detected by real time RT-PCR, and Western blot and immunofluorescence were applied. The expression level of closely linked related proteins, Claudin-1, Claudin-3, Occludin and ZO-1, was detected by light staining. In order to verify the protective effect of IL-17A, the embedded Transwell was used to cultivate Caco-2 cells, to construct a cell monolayer in vitro, and to observe the gp160 and Tat of the SHIV/SIV protein component and the IL-17 on the epithelial cell barrier across the epithelium. The effect of cell resistance, FITC-Dextran permeability, and the use of real time RT-PCR to detect the transcriptional level of claudin-1, Claudin-3 and ZO-1 genes associated with close connections in cell monolayers. The study found that the proportion of CD3+CD4~+T cells in the mucosa of the digestive tract of SIV/SHIV infected animals was lower than that in the non infected animals; IFN gamma +CD3+CD4~+T cells and IL-4+CD3+CD4~+. The proportion of T cells was higher than that of the uninfected animals. The transcriptional level of Th1 cells and Th2 nuclear transcription factors T-bet and GATA-3 was higher than that of the uninfected animals. In the intestinal mucosal tissue of the virus infection group, the proportion of IL-17+CD3+CD4~+T cells was lower than the ileum and the proportion of IL-17+CD3+CD4~+T cells was lower than that of the normal group. The three groups were infected with the transcription of IL-17 in the digestive tract mucosa of the animals. The levels of IL-10+CD3+CD4~+ T cells and Foxp3+CD3+CD4~+ T cells were lower than those of normal animals, and the proportion of Foxp3+CD3+CD4~+T cells in SIV/SHIV infected animals was lower than that of normal animals. The proportion of these two kinds of cells in the other digestive tract mucosa was higher than that of normal animals; real time RT-PCR detection Foxp3 showed that except the Gr4 group jejunum, the real time RT-PCR detection Foxp3 showed that the proportion of the cells was higher than that of the normal animals. The Foxp3 transcriptional level of the cecum and colon was lower than that of the normal animal. The Foxp3 transcriptional level in the digestive tract of the other two groups of SHIV infected animals was higher than that of the normal animals. In the process of studying the epithelial barrier of the digestive tract of the animals, the ultrastructure of the small intestine and the epithelial cells of the large intestine in the group Gr4 and the Gr19 group were observed by transmission electron microscope. Microstructures, compared with normal animals, can be seen that the close connection between the epithelial cells widened and the gap between the cells increased: microvilli arranged and scattered, thinning, thinning, appearing in lodging, partly broken and falling off; the endoplasmic reticulum has dilatation, the mitochondria swollen, and the vacuoles appear in the cytoplasm. At gene level, the transcription level of Claudin-1 in the infected animals Compared with the normal group, the transcriptional level of Claudin-1 in the Gr19 group of the rectum was lower than that in the normal group; the protein level, the protein expression level of Claudin-1 in the jejunum, the cecum and the colon mucosa of the infected animals was lower than that of the normal group. The distribution of Claudin-1 in the digestive tract mucosa of the virus infected animals and the eggs were observed by the confocal laser scanning microscope. The level of white expression was affected, distribution was scattered,.Claudin-3 at low fluorescence was low at gene level, and the transcriptional level of Claudin-3 in infected animals duodenum and jejunum was lower than that in normal group, and the transcription level of Claudin-3 in Gr19 group of infected animal ileum, colon and rectum was lower than that in normal group; protein level infected the jejunum, colon and straight of animals. The protein expression level of Claudin-3 in the intestinal mucosa was lower than that in the normal group, and the expression level of Claudin-3 in the Gr19 group of the duodenum was higher than that in the normal group, and the Gr15 group decreased. The protein expression level of Claudin-3 in the Gr19 and Gr15 groups in the cecum was lower than that in the normal group. The.Real-time RT PCR detection was found in the occludin of the duodenum, jejunum and rectum in the infected animals. Western blot detection showed that the protein expression level of Occludin in the intestinal mucosa of infected animals was lower than that of the normal group. The protein expression level of Occludin in the Gr19 and Gr4 groups in the colon was lower than that in the normal group. The transcriptional level of ZO-1 in the duodenum and jejunum of the infected animals was lower than that in the normal group. The distribution of ZO-1 and the level of protein expression in the digestive tract mucosa of the animals were affected by the laser confocal microscopy. We found that the distribution was discontinuous and the fluorescence degree was reduced. We found that we could establish stable Caco-2 cells in vitro in vitro. System, Tat (14ng/ml) has immediate effect on cell monolayer, that is, 2h will increase the permeability of cell monolayer and reduce TER; specific concentration Tat and GP1 60 have little effect on the expression of closely linked genes in Caco-2 cell monolayer; IL-17 (100ng/ml) has a certain protective effect on cell monolayer. To sum up, SIV/SHIV infection is constant. The proportion of CD3+CD4~+ T cells and IL-17+CD3+CD4~+T cells in the mucous membrane of the river monkey decreased. The transcriptional level of the closely linked claudin-1, Claudin-3, occludin and ZO-1 genes and the expression level of the related proteins decreased in varying degrees. Tat (14ng/ml) had immediate effect on the monolayer of cells, and IL-17 (100ng/ml) was a single layer of cell monolayers. These results have a certain degree of protective effect, and these results provide new ideas for the discovery of new therapeutic strategies, and explore new ways to reduce microbial translocation and immune activation.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R512.91
[Abstract]:The loss of CD4~+T cells and the continuous immune activation of the body are the main immuno pathological features of HIV infection. Virus infection leads to damage of the structure and function of the intestinal immune system, the destruction of the mucosal barrier, and the translocation of microorganisms, which may lead to the chronic immune activation of the body. This may be an important mechanism for the pathogenesis of HIV-1. Although Th17 cells are infected during the HIV infection process. The effect of the loss of the cells on the immune system is not clear, especially the role of Th17 cells in the mucosal barrier damage. In this paper, the changes and relationships of the CD4~+T Th17 cells in the digestive tract mucosa and the mucosal barrier structure in the digestive tract were analyzed by SIV/SHIV infection in Ganges RIver monkey. At the same time, Ca was applied to the infection of the mucosal barrier. The cell monolayers of CO-2 cells were constructed in vitro, and the effects of SIV/SHIV protein components gp160 and Tat and IL-17 on the epithelial barrier were provided to provide new ideas for the repair of mucosal barrier damage caused by HIV-1 infection. In vivo, in order to understand the effect of SIV/SHIV infection on the mucous membrane CD4~+TTh17 cells of the digestive tract of Ganges RIver monkey, the flow finer was used in this paper. Cytoplasm was used to detect the relative size of CD3+CD4~+T, IL-17+CD3+CD4~+T, IL-10+CD3+CD4~+T, Foxp3+CD3+CD4~+T, IL-4+CD3+CD4~+T and IFNy+CD3+CD4~+T cells, and the expression levels of IL-17A, RORyt, TGF- beta, GATA-3, T-bet, and IFNy+CD3+CD4~+T were detected by realtimeRT-PCR. In order to understand the changes in the mucosal barrier after SIV/SHIV infection, the ultrastructure of the mucosal epithelium in the digestive tract of normal and infected animals was observed by transmission electron microscopy. The transcriptional level of the claudin-1, Claudin-3, occludin and ZO-1 genes related to the close connection was detected by real time RT-PCR, and Western blot and immunofluorescence were applied. The expression level of closely linked related proteins, Claudin-1, Claudin-3, Occludin and ZO-1, was detected by light staining. In order to verify the protective effect of IL-17A, the embedded Transwell was used to cultivate Caco-2 cells, to construct a cell monolayer in vitro, and to observe the gp160 and Tat of the SHIV/SIV protein component and the IL-17 on the epithelial cell barrier across the epithelium. The effect of cell resistance, FITC-Dextran permeability, and the use of real time RT-PCR to detect the transcriptional level of claudin-1, Claudin-3 and ZO-1 genes associated with close connections in cell monolayers. The study found that the proportion of CD3+CD4~+T cells in the mucosa of the digestive tract of SIV/SHIV infected animals was lower than that in the non infected animals; IFN gamma +CD3+CD4~+T cells and IL-4+CD3+CD4~+. The proportion of T cells was higher than that of the uninfected animals. The transcriptional level of Th1 cells and Th2 nuclear transcription factors T-bet and GATA-3 was higher than that of the uninfected animals. In the intestinal mucosal tissue of the virus infection group, the proportion of IL-17+CD3+CD4~+T cells was lower than the ileum and the proportion of IL-17+CD3+CD4~+T cells was lower than that of the normal group. The three groups were infected with the transcription of IL-17 in the digestive tract mucosa of the animals. The levels of IL-10+CD3+CD4~+ T cells and Foxp3+CD3+CD4~+ T cells were lower than those of normal animals, and the proportion of Foxp3+CD3+CD4~+T cells in SIV/SHIV infected animals was lower than that of normal animals. The proportion of these two kinds of cells in the other digestive tract mucosa was higher than that of normal animals; real time RT-PCR detection Foxp3 showed that except the Gr4 group jejunum, the real time RT-PCR detection Foxp3 showed that the proportion of the cells was higher than that of the normal animals. The Foxp3 transcriptional level of the cecum and colon was lower than that of the normal animal. The Foxp3 transcriptional level in the digestive tract of the other two groups of SHIV infected animals was higher than that of the normal animals. In the process of studying the epithelial barrier of the digestive tract of the animals, the ultrastructure of the small intestine and the epithelial cells of the large intestine in the group Gr4 and the Gr19 group were observed by transmission electron microscope. Microstructures, compared with normal animals, can be seen that the close connection between the epithelial cells widened and the gap between the cells increased: microvilli arranged and scattered, thinning, thinning, appearing in lodging, partly broken and falling off; the endoplasmic reticulum has dilatation, the mitochondria swollen, and the vacuoles appear in the cytoplasm. At gene level, the transcription level of Claudin-1 in the infected animals Compared with the normal group, the transcriptional level of Claudin-1 in the Gr19 group of the rectum was lower than that in the normal group; the protein level, the protein expression level of Claudin-1 in the jejunum, the cecum and the colon mucosa of the infected animals was lower than that of the normal group. The distribution of Claudin-1 in the digestive tract mucosa of the virus infected animals and the eggs were observed by the confocal laser scanning microscope. The level of white expression was affected, distribution was scattered,.Claudin-3 at low fluorescence was low at gene level, and the transcriptional level of Claudin-3 in infected animals duodenum and jejunum was lower than that in normal group, and the transcription level of Claudin-3 in Gr19 group of infected animal ileum, colon and rectum was lower than that in normal group; protein level infected the jejunum, colon and straight of animals. The protein expression level of Claudin-3 in the intestinal mucosa was lower than that in the normal group, and the expression level of Claudin-3 in the Gr19 group of the duodenum was higher than that in the normal group, and the Gr15 group decreased. The protein expression level of Claudin-3 in the Gr19 and Gr15 groups in the cecum was lower than that in the normal group. The.Real-time RT PCR detection was found in the occludin of the duodenum, jejunum and rectum in the infected animals. Western blot detection showed that the protein expression level of Occludin in the intestinal mucosa of infected animals was lower than that of the normal group. The protein expression level of Occludin in the Gr19 and Gr4 groups in the colon was lower than that in the normal group. The transcriptional level of ZO-1 in the duodenum and jejunum of the infected animals was lower than that in the normal group. The distribution of ZO-1 and the level of protein expression in the digestive tract mucosa of the animals were affected by the laser confocal microscopy. We found that the distribution was discontinuous and the fluorescence degree was reduced. We found that we could establish stable Caco-2 cells in vitro in vitro. System, Tat (14ng/ml) has immediate effect on cell monolayer, that is, 2h will increase the permeability of cell monolayer and reduce TER; specific concentration Tat and GP1 60 have little effect on the expression of closely linked genes in Caco-2 cell monolayer; IL-17 (100ng/ml) has a certain protective effect on cell monolayer. To sum up, SIV/SHIV infection is constant. The proportion of CD3+CD4~+ T cells and IL-17+CD3+CD4~+T cells in the mucous membrane of the river monkey decreased. The transcriptional level of the closely linked claudin-1, Claudin-3, occludin and ZO-1 genes and the expression level of the related proteins decreased in varying degrees. Tat (14ng/ml) had immediate effect on the monolayer of cells, and IL-17 (100ng/ml) was a single layer of cell monolayers. These results have a certain degree of protective effect, and these results provide new ideas for the discovery of new therapeutic strategies, and explore new ways to reduce microbial translocation and immune activation.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R512.91
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