HIV感染中Th17细胞与消化道上皮屏障完整性关系的实验研究
发布时间：2018-07-13 17:49
【摘要】：CD4~+T细胞缺失和机体持续的免疫激活是HIV感染的主要免疫病理特征。病毒感染导致肠道免疫系统结构和功能的损伤、粘膜屏障破坏、微生物易位,从而导致机体慢性免疫活化,这可能是HIV-1致病的重要机制。虽然HIV感染过程中Th17细胞发生显著变化,但该细胞的缺失对免疫系统的影响尚不清楚,尤其是Th17细胞在粘膜屏障损伤中的作用亟待研究。本文应用SIV/SHIV感染恒河猴分析消化道粘膜CD4~+T Th17细胞与消化道粘膜屏障结构在病毒感染后的变化及相互关系;同时应用Caco-2细胞体外构建细胞单层,探讨SIV/SHIV蛋白组分gp160和Tat以及IL-17对上皮细胞屏障的影响,为HIV-1感染所致粘膜屏障损伤的修复提供新思路。在体内实验研究中,为了解SIV/SHIV感染对恒河猴消化道粘膜CD4~+TTh17细胞的影响,本文采用流式细胞术检测了 CD3+CD4~+T细胞、IL-17+CD3+CD4~+T细胞、IL-10+CD3+CD4~+T 细胞、Foxp3+CD3+CD4~+T 细胞、IL-4+CD3+CD4~+T 细胞和IFNy+CD3+CD4~+T细胞亚群的相对大小;并通过realtimeRT-PCR检测了IL-17A、RORyt、TGF-β、GATA-3、T-bet和Foxp3等基因mRNA表达水平在病毒感染后的变化。为了解粘膜屏障在SIV/SHIV感染后的变化,本文利用透射电子显微镜观察了正常动物和感染动物消化道粘膜上皮的超微结构,采用real time RT-PCR检测了与紧密连接相关的claudin-1,claudin-3,occludin和ZO-1基因的转录水平,应用western blot和免疫荧光染色技术检测了紧密连接相关的相关蛋白Claudin-1,Claudin-3,Occludin和ZO-1的表达水平。为验证IL-17A的保护作用,本文采用嵌入式transwell培养Caco-2细胞,体外构建细胞单层,观察了 SHIV/SIV蛋白组分gp160和Tat以及IL-17对上皮细胞屏障跨上皮细胞电阻、FITC-Dextran通透性的影响,同时应用real time RT-PCR检测了细胞单层中与紧密连接相关的claudin-1,claudin-3和ZO-1基因的转录水平。研究发现,SIV/SHIV感染动物消化道粘膜中CD3+CD4~+T细胞比例较未感染动物低;IFNγ+CD3+CD4~+T细胞和IL-4+CD3+CD4~+T细胞比例较未感染动物的高,Th1细胞和Th2细胞核转录因子T-bet和GATA-3的转录水平与未感染动物相比有不同程度的升高;在病毒感染组肠道粘膜组织中,除回肠,IL-17+CD3+CD4~+T细胞比例较正常组降低,三组感染动物消化道粘膜中IL-17的转录水平均低于正常动物;检测 IL-10+CD3+CD4~+ T 细胞和 Foxp3+CD3+CD4~+ T 细胞,SIV/SHIV感染动物除结肠Foxp3+CD3+CD4~+T细胞比例较正常动物降低,其余消化道粘膜组织中这两类细胞比例较正常动物均升高;real time RT-PCR检测Foxp3结果显示,除Gr4组空肠、盲肠和结肠Foxp3转录水平与正常动物相比较降低,另两组SHIV感染动物消化道粘膜组织中Foxp3转录水平与正常动物相比较高。在研究动物消化道上皮屏障的过程中,通过透射电子显微镜观察Gr4组和Gr19组动物小肠和大肠粘膜上皮细胞超微结构,与正常动物进行对比,可见上皮细胞间紧密连接增宽,胞间缝隙增大:微绒毛排列散乱、变细、稀疏,呈现倒伏状,有部分断裂、脱落;内质网有扩张现象,线粒体有肿胀,在胞质内有空泡出现。在基因水平,感染动物中claudin-1的转录水平较正常组均降低,直肠中Gr19组claudin-1的转录水平较正常组降低;蛋白水平,感染组动物的空肠、盲肠和结肠粘膜中Claudin-1的蛋白表达水平较正常组降低,激光共聚焦显微镜观察发现病毒感染组动物消化道粘膜组织中Claudin-1的分布和蛋白表达水平均受到影响,分布散乱、荧光度低。Claudin-3在基因水平,感染动物十二指肠和空肠claudin-3的转录水平较正常组均降低;感染动物回肠、结肠和直肠中Gr19组claudin-3的转录水平较正常组均降低;蛋白水平感染动物的空肠、结肠和直肠粘膜中Claudin-3的蛋白表达水平较正常组降低;十二指肠中Gr19组Claudin-3的表达水平较正常组升高,Gr15组降低;盲肠中Gr19组和Gr15组Claudin-3的蛋白表达水平较正常组降低。Real-time RT PCR检测发现在感染动物十二指肠、空肠和直肠occludin的转录水平较正常组均降低;western blot检测发现感染动物小肠粘膜中Occludin的蛋白表达水平均较低,结肠中Gr19组和Gr4组Occludin的蛋白表达水平较正常组降低。Real-timeRTPCR检测发现在感染动物十二指肠和空肠ZO-1的转录水平较正常组均降低;激光共聚焦显微镜观察动物消化道粘膜组织免疫荧光染色切片,发现病毒感染组动物消化道粘膜组织中ZO-1的分布和蛋白表达水平均受到影响,分布不连续、荧光度降低。在体外实验研究中发现,我们可以建立稳定的Caco-2细胞体外研究体系,Tat(14ng/ml)对细胞单层具有即时影响效应,即作用2h后会增加细胞单层通透性并降低TER;特定浓度Tat和gp1 60对Caco-2细胞单层紧密连接相关基因表达的影响不大;IL-17(100ng/ml)对细胞单层有一定程度的保护作用。综上所述,SIV/SHIV感染恒河猴消化道粘膜CD3+CD4~+ T细胞和IL-17+CD3+CD4~+T 细胞比例下降,与紧密连接相关的 claudin-1,claudin-3,occludin和ZO-1基因的转录水平以及相关蛋白的表达水平有不同程度的下降,Tat(14ng/ml)对细胞单层具有即时影响效应,IL-17(100ng/ml)对细胞单层有一定程度的保护作用,这些结果为新的治疗策略的发现、探索新的方法减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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R512.91
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