HIV-1感染对肠粘膜回归相关基因表达影响的实验研究
发布时间：2018-08-12 17:50
【摘要】：目的：HIV-1病毒感染引起粘膜CD4+T细胞大量缺失,导致粘膜免疫功能损伤、微生物易位及慢性免疫激活,最终导致全身免疫缺陷发展成为AIDS。虽然感染早期粘膜CD4+T细胞大量感染,CD4+T淋巴细胞数显著下降,但是外周血CD4+T淋巴细胞在感染后却长期维持在比较高的水平。粘膜CD4+T淋巴细胞数量减少的原因目前尚未完全清楚。除了病毒感染直接或间接杀伤CD4+T淋巴细胞外,淋巴细胞分布异常也可能是一些组织中CD4+T淋巴细胞减少的原因。淋巴细胞循环是淋巴细胞向特定组织迁移的过程,由多种分子参与,涉及选择素家族、整合素家族、免疫球蛋白超家族的粘附分子和多种细胞因子。对这些分子在HIV-1感染后的变化尚无系统研究。本研究以SIV/SHIV感染恒河猴为动物模型,探索HIV-1对免疫细胞回归相关基因的影响。 方法：1)提取恒河猴结肠组织总RNA,利用5'RACE和3'RACE分别扩增cDNA序列,将获得的序列构建到pGEM-Teasy载体测序,利用DNAMan软件进行序列比对和拼接获得MAdCAM-1cDNA全序列：从GenBank上获得40多个物种MAdCAM-1核苷酸序列,利用BioEidt和MEGA软件进行比对和构建系统进化树。2)根据天根RNA提取试剂盒说明提取组织RNA、根据康为世纪组织蛋白抽提试剂盒说明提取组织蛋白,利用半定量RT-PCR方法和实时荧光定量RT-PCR方法检测组织中MAdCAM-1mRNA的表达水平,并用Western Blot方法检测了十二指肠活检组织中MAdCAM-1蛋白的表达水平。另外利用RT-PCR方法检测MAdCAM-1exon4缺失的剪切变异体。3)基因合成恒河猴MAdCAM-1编码区全序列(MFL)和第四个外显子缺失剪切变异体(ML4),通过酶切连接,构建到pCDNA3.1真核表达载体上,转染293F细胞,利用流式细胞术检测细胞表面MAdCAM-1分子的表达。将转染后293F细胞用CFSE染色,Hut-78细胞用PKH26染色,在Mn2+存在下进行粘附实验。4)选取正常未感染恒河猴和SIV/SHIV感染的Gr4组、SHIV感染的Gr15组和Gr19组恒河猴冻存组织样本(感染动物样品主要来自本实验室以往感染实验的病毒对照组),按照试剂盒说明提取组织RNA,建立多种基因实时荧光定量RT-PCR检测方法,用以检测MAdCAM-1、ITGA4、ITGB7、NKX2.3、CCL25、 CCR9、CCL28、CCR10、CCL19、CCL21、CCR7、RGS1、CCL22、CCR4、CCL20、CCR6、 GPR183、FCGRT、PIGR、BCL-6、DDIT3、TNF-α、IFN-γ等23种基因的转录水平。5)按照康为世纪组织蛋白抽提试剂盒说明提取四组动物组织蛋白,利用Western Blot检测MAdCAM-1组织蛋白表达；利用ELISA检测动物组织CCL20、TNF-α、IFN-γ、IL-17、IL-6等因子的蛋白表达水平。 结果:MAdCAM-1在粘膜特异回归中具有重要作用。本文首先克隆了恒河猴MAdCAM-1基因,研究了该基因的组织分布与感染后的变化。发现恒河猴MAdCAM-1cDNA核苷酸序列全长为1503bp,包括一个14bp的5’非编码区(5'UTR)和一个403bp的3’非编码区(3’UTR),在1463bp位置有一个AATAAA的多聚加尾信号。推测的恒河猴MAdCAM-1蛋白与人MAdCAM-1相似,含有一个信号肽,两个N端Ig样结构域,一个粘蛋白样结构域,一个跨膜区和一个胞浆区,但是没有小鼠中的第三个Ig样结构域和IgA1样结构域。与a4β7结合的LDT保守区在第一个Ig样结构域内。 在正常恒河猴组织中,MAdCAM-1mRNA主要表达于胃肠道、脾和淋巴结中,在肠系膜淋巴结中表达量最高。在肠道中,大肠中表达量高于小肠。皮肤、胸腺、口腔粘膜和肝脏中MAdCAM-1mRNA的表达水平很低,其中肝脏中的表达量最低。在SHIV感染后的十二指肠粘膜组织中,MAdCAM-1的mRNA表达水平显著下降,MAdCAM-1蛋白表达水平也下降。 为进一步了解SIV/SHIV感染对淋巴归巢相关基因表达的影响,本文研究了MAdACM-1及其受体α4β7在正常和感染动物肠粘膜中的表达水平。发现SIV/SHIV感染后MAdCAM-1mRNA在小肠中下降大肠中上升,同时MAdCAM-1蛋白.ITGA4、ITGB7和NKX2.3基因表达也呈现同样的变化趋势。在正常肠粘膜组织中MAdCAM-1、ITGA4、ITGB7的mRNA之间的分布具有相关性,而MAdCAM-1与NKX2.3之间的分布不具有相关性,但感染后NKX2.3与MAdCAM-1在肠道分布相关,可能参与MAdCAM-1在肠道的表达调控。 更进一步研究发现SIV/SHIV感染也影响了消化道粘膜中多种趋化因子及受体转录水平。其中,感染后肠道相关趋化因子CCL25和CCL28及其受体在小肠中呈下降趋势,而在大肠中呈上升趋势,不同病毒感染影响也不同。感染后趋化因子CCL19、CCL21和受体CCR7mRNA在淋巴器官没有明显变化,但在十二指肠的表达显著下降；趋化因子CCL20mRNA的表达在感染后没有显著变化,而受体CCR6mRNA在十二指肠和空肠显著下降；感染后趋化因子CCL22mRNA在空肠下降,受体CCR4mRNA在腹股沟淋巴结中下降。 另外,SIV/SHIV感染也影响细胞迁移调节基因在转录水平的表达：GPR183调节B细胞在淋巴滤泡中的迁移,GPRI83mRNA在脾、肠系膜淋巴结和腹股沟淋巴结中的表达显著降低,RGS1mRNA在胃底和十二指肠的表达降低。 为了解回归相关分子的变化与粘膜中其他免疫病理变化的关系,本文研究了SIV/SHIV感染后炎症因子TNF-α、IFN-γ的转录水平的变化。发现总体上TNF-α mRNA表达量在小肠前段下降,从回肠到直肠上升；IFN-γ mRNA在直肠显著降低,其他部位没有明显变化。炎症因子TNF-α蛋白在空肠和回肠显著下降,整体上呈现在小肠中下降在大肠中上升的趋势；IFN-γ蛋白没有明显变化、IL-6蛋白在回肠和盲肠显著上升、IL-17蛋白在直肠显著上升。另外还观察了与IgA转运相关基因转录水平变化,发现FCGRT mRNA在肠道粘膜表达水平上升,而PIGR mRNA在胃肠道和淋巴结表达下降,SIV/SHIV感染后BCL-6mRNA在小肠中下降,大肠中上升,而SHIV感染后BCL-6mRNA在胃肠道粘膜(除直肠外)、脾和淋巴结中均下降。以上结果表明,SIV/SHIV感染后粘膜体液免疫功能可能受到了影响。 结论：本研究1)获得了恒河猴MAdCAM-1cDNA核苷酸序列,发现与人MAdCAM-1序列高度相似；2)发现SIV/SHIV感染影响粘膜回归相关粘附分子MAdCAM-1与受体α4β7的表达；3)发现SIV/SHIV感染影响多种粘膜回归相关趋化因子与受体的表达；4)发现粘膜回归相关因子在SIV/SHIV感染后的变化与粘膜内炎症水平相关；5)发现粘膜回归相关分子在SIV/SHIV感染后的改变可能伴随着粘膜体液免疫功能的变化。这些结果表明HIV-1可能通过影响粘膜淋巴细胞回归,进而影响粘膜细胞和体液免疫功能,为深入认识HIV/AIDS病理过程和寻找新的生物学干预靶点提供了重要基础。
[Abstract]:AIM: Mucosal CD4 + T cell deletion, microbial translocation and chronic immune activation caused by HIV-1 virus infection eventually lead to the development of systemic immunodeficiency into AIDS. The cause of the decrease in the number of CD4 + T lymphocytes in the mucosa is not fully understood. In addition to the direct or indirect killing of CD4 + T lymphocytes by virus infection, the abnormal distribution of lymphocytes may also be responsible for the decrease of CD4 + T lymphocytes in some tissues. Lymphocyte circulation is the cause of lymphocyte turnover to specific groups. Tissue migration involves multiple molecules, including selectin family, integrin family, immunoglobulin superfamily adhesion molecules and cytokines. The changes of these molecules after HIV-1 infection have not been systematically studied. Influence.
METHODS: 1) Total RNA was extracted from colon tissue of rhesus monkey, and the sequences were amplified by 5'RACE and 3'RACE respectively. The obtained sequences were constructed into pGEM-Teasy vector and sequenced. The complete sequence of MAdCAM-1 was obtained by DNA Man software. More than 40 species of MAdCAM-1 nucleotide sequences were obtained from GenBank, and BioEidt and MEGA software were used. Comparing and constructing phylogenetic tree.2) Extracting tissue RNA according to Tiangen RNA extraction kit, extracting tissue protein according to Kangwei Century Tissue Protein Extraction kit, using semi-quantitative RT-PCR and real-time fluorescence quantitative RT-PCR to detect the expression of MAdCAM-1 mRNA in tissues, and Western Blot method to detect the expression of MAdCAM-1 mRNA. The expression level of MAdCAM-1 protein in duodenal biopsy tissues was detected by RT-PCR. In addition, the splicing variant of MAdCAM-1 exon 4 deletion was detected by RT-PCR. 3) gene was synthesized into the full sequence of MAdCAM-1 coding region (MFL) and the fourth exon deletion splicing variant (ML4) of rhesus monkey, and then constructed into pCDNA3.1 eukaryotic expression vector by enzyme digestion ligation and transfected 293F fine. After transfection, 293F cells were stained with CFSE, and Hut-78 cells were stained with PKH26. Adhesion test was carried out in the presence of Mn2 +. 4. Gr4, Gr15 and Gr19, which were not infected with SIV / SHIV, were selected as the control group. According to the instructions of the kit, RNA was extracted from the tissues and real-time fluorescence quantitative RT-PCR was used to detect MAdCAM-1, ITGA4, ITGB7, NKX2.3, CCL25, CCR9, CCL28, CCR10, CCL19, CCL21, CCR7, RGS1, CCL22, CCR4, CCL20, CCR6, GPR183, FCGRT, PIGR, BCL-6, DDIT3, CCL21, CCL21, CCR7, CCR7, CCR7, R7, RGS1, CCL22, CCR4, CCL20, CCR6, GPR183, FCGRT, PIGR, BC Transcription levels of 23 genes, such as - alpha, IFN - gamma and so on. 5) According to Kangwei Century Tissue Protein Extraction Kit, four groups of animal tissue proteins were extracted, and the expression of MAdCAM-1 was detected by Western Blot, and the expression levels of CCL20, TNF-alpha, IFN-gamma, IL-17 and IL-6 were detected by ELISA.
Results: MAdCAM-1 plays an important role in mucosa-specific regression. Firstly, the MAdCAM-1 gene of rhesus monkey was cloned and its tissue distribution and changes after infection were studied. The hypothetical MAdCAM-1 protein of rhesus monkey, similar to human MAdCAM-1, contains a signal peptide, two N-terminal Ig-like domains, one mucin-like domain, one transmembrane domain and one cytoplasmic domain, but does not bind to the third Ig-like domain and IgA1-like domain in mice. The conserved region of LDT is in the first Ig like domain.
In normal rhesus monkey tissues, MAdCAM-1 mRNA was mainly expressed in gastrointestinal tract, spleen and lymph nodes, and was the highest in mesenteric lymph nodes. In intestine, the expression level of MAdCAM-1 mRNA was higher in large intestine than in small intestine, skin, thymus, oral mucosa and liver. The expression level of MAdCAM-1 mRNA in liver was the lowest. The expression level of MAdCAM-1 mRNA and MAdCAM-1 protein in intestinal mucosa decreased significantly.
To further understand the effect of SIV/SHIV infection on the expression of lymphatic homing-related genes, the expression of MAdACM-1 and its receptor alpha 4 beta-7 in intestinal mucosa of normal and infected animals was studied. The distribution of MAdCAM-1, ITGA4 and ITGB7 mRNA in normal intestinal mucosa was correlated, but the distribution of MAdCAM-1 and NKX2.3 was not correlated, but NKX2.3 was correlated with the distribution of MAdCAM-1 in intestine after infection, which may be involved in the regulation of MAdCAM-1 expression in intestine.
Further studies showed that SIV/SHIV infection also affected the levels of chemokines and receptors in gastrointestinal mucosa. After infection, intestinal chemokines CCL25, CCL28 and their receptors decreased in the small intestine, but increased in the large intestine, and the effects of different viral infections were different. The expression of chemokine CCL20 mRNA did not change significantly after infection, whereas the expression of chemokine CCR6 mRNA in duodenum and jejunum decreased significantly; the expression of chemokine CCL22 mRNA in jejunum decreased significantly after infection, and the expression of chemokine CCR4 mRNA in inguinal lymph nodes decreased significantly. Drop.
In addition, SIV/SHIV infection also affected the expression of cell migration regulator genes at transcriptional level: GPR183 regulated the migration of B cells in lymphoid follicles, decreased the expression of GPRI83 mRNA in spleen, mesenteric and inguinal lymph nodes, and decreased the expression of RGS1 mRNA in gastric fundus and duodenum.
In order to understand the relationship between the changes of regression-related molecules and other immunopathological changes in mucosa, we studied the changes of the transcription levels of inflammatory factors TNF-a and IFN-gamma after SIV/SHIV infection. Inflammatory factor TNF-alpha protein decreased significantly in jejunum and ileum, but decreased in small intestine and increased in large intestine as a whole; IFN-gamma protein did not change significantly, IL-6 protein increased significantly in ileum and cecum, and IL-17 protein increased significantly in rectum. It was found that FCGRT mRNA increased in intestinal mucosa, PIGR mRNA decreased in gastrointestinal tract and lymph nodes, BCL-6 mRNA decreased in small intestine and increased in large intestine after SIV/SHIV infection, while BCL-6 mRNA decreased in gastrointestinal mucosa (except rectum), spleen and lymph nodes after SHIV infection. The function of the disease may be affected.
CONCLUSIONS: In this study, the sequence of MAdCAM-1 cDNA in rhesus monkeys was obtained and found to be highly similar to that of human MAdCAM-1; 2) SIV/SHIV infection was found to affect the expression of MAdCAM-1 and receptor alpha 4 beta 7; 3) SIV/SHIV infection was found to affect the expression of multiple mucosal regression-related chemokines and receptors; 4) SIV/SHIV infection was found to affect the expression of multiple mucosal regression-related chemokines and receptors These results suggest that HIV-1 may affect mucosal and humoral immune function by influencing mucosal lymphocyte regression, and then influence mucosal cells and humoral immune function. It provides an important basis for understanding the pathological process of HIV/AIDS and finding new biological intervention targets.
【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】：2014
【分类号】：R512.91
，
本文编号：2179831
[Abstract]:AIM: Mucosal CD4 + T cell deletion, microbial translocation and chronic immune activation caused by HIV-1 virus infection eventually lead to the development of systemic immunodeficiency into AIDS. The cause of the decrease in the number of CD4 + T lymphocytes in the mucosa is not fully understood. In addition to the direct or indirect killing of CD4 + T lymphocytes by virus infection, the abnormal distribution of lymphocytes may also be responsible for the decrease of CD4 + T lymphocytes in some tissues. Lymphocyte circulation is the cause of lymphocyte turnover to specific groups. Tissue migration involves multiple molecules, including selectin family, integrin family, immunoglobulin superfamily adhesion molecules and cytokines. The changes of these molecules after HIV-1 infection have not been systematically studied. Influence.
METHODS: 1) Total RNA was extracted from colon tissue of rhesus monkey, and the sequences were amplified by 5'RACE and 3'RACE respectively. The obtained sequences were constructed into pGEM-Teasy vector and sequenced. The complete sequence of MAdCAM-1 was obtained by DNA Man software. More than 40 species of MAdCAM-1 nucleotide sequences were obtained from GenBank, and BioEidt and MEGA software were used. Comparing and constructing phylogenetic tree.2) Extracting tissue RNA according to Tiangen RNA extraction kit, extracting tissue protein according to Kangwei Century Tissue Protein Extraction kit, using semi-quantitative RT-PCR and real-time fluorescence quantitative RT-PCR to detect the expression of MAdCAM-1 mRNA in tissues, and Western Blot method to detect the expression of MAdCAM-1 mRNA. The expression level of MAdCAM-1 protein in duodenal biopsy tissues was detected by RT-PCR. In addition, the splicing variant of MAdCAM-1 exon 4 deletion was detected by RT-PCR. 3) gene was synthesized into the full sequence of MAdCAM-1 coding region (MFL) and the fourth exon deletion splicing variant (ML4) of rhesus monkey, and then constructed into pCDNA3.1 eukaryotic expression vector by enzyme digestion ligation and transfected 293F fine. After transfection, 293F cells were stained with CFSE, and Hut-78 cells were stained with PKH26. Adhesion test was carried out in the presence of Mn2 +. 4. Gr4, Gr15 and Gr19, which were not infected with SIV / SHIV, were selected as the control group. According to the instructions of the kit, RNA was extracted from the tissues and real-time fluorescence quantitative RT-PCR was used to detect MAdCAM-1, ITGA4, ITGB7, NKX2.3, CCL25, CCR9, CCL28, CCR10, CCL19, CCL21, CCR7, RGS1, CCL22, CCR4, CCL20, CCR6, GPR183, FCGRT, PIGR, BCL-6, DDIT3, CCL21, CCL21, CCR7, CCR7, CCR7, R7, RGS1, CCL22, CCR4, CCL20, CCR6, GPR183, FCGRT, PIGR, BC Transcription levels of 23 genes, such as - alpha, IFN - gamma and so on. 5) According to Kangwei Century Tissue Protein Extraction Kit, four groups of animal tissue proteins were extracted, and the expression of MAdCAM-1 was detected by Western Blot, and the expression levels of CCL20, TNF-alpha, IFN-gamma, IL-17 and IL-6 were detected by ELISA.
Results: MAdCAM-1 plays an important role in mucosa-specific regression. Firstly, the MAdCAM-1 gene of rhesus monkey was cloned and its tissue distribution and changes after infection were studied. The hypothetical MAdCAM-1 protein of rhesus monkey, similar to human MAdCAM-1, contains a signal peptide, two N-terminal Ig-like domains, one mucin-like domain, one transmembrane domain and one cytoplasmic domain, but does not bind to the third Ig-like domain and IgA1-like domain in mice. The conserved region of LDT is in the first Ig like domain.
In normal rhesus monkey tissues, MAdCAM-1 mRNA was mainly expressed in gastrointestinal tract, spleen and lymph nodes, and was the highest in mesenteric lymph nodes. In intestine, the expression level of MAdCAM-1 mRNA was higher in large intestine than in small intestine, skin, thymus, oral mucosa and liver. The expression level of MAdCAM-1 mRNA in liver was the lowest. The expression level of MAdCAM-1 mRNA and MAdCAM-1 protein in intestinal mucosa decreased significantly.
To further understand the effect of SIV/SHIV infection on the expression of lymphatic homing-related genes, the expression of MAdACM-1 and its receptor alpha 4 beta-7 in intestinal mucosa of normal and infected animals was studied. The distribution of MAdCAM-1, ITGA4 and ITGB7 mRNA in normal intestinal mucosa was correlated, but the distribution of MAdCAM-1 and NKX2.3 was not correlated, but NKX2.3 was correlated with the distribution of MAdCAM-1 in intestine after infection, which may be involved in the regulation of MAdCAM-1 expression in intestine.
Further studies showed that SIV/SHIV infection also affected the levels of chemokines and receptors in gastrointestinal mucosa. After infection, intestinal chemokines CCL25, CCL28 and their receptors decreased in the small intestine, but increased in the large intestine, and the effects of different viral infections were different. The expression of chemokine CCL20 mRNA did not change significantly after infection, whereas the expression of chemokine CCR6 mRNA in duodenum and jejunum decreased significantly; the expression of chemokine CCL22 mRNA in jejunum decreased significantly after infection, and the expression of chemokine CCR4 mRNA in inguinal lymph nodes decreased significantly. Drop.
In addition, SIV/SHIV infection also affected the expression of cell migration regulator genes at transcriptional level: GPR183 regulated the migration of B cells in lymphoid follicles, decreased the expression of GPRI83 mRNA in spleen, mesenteric and inguinal lymph nodes, and decreased the expression of RGS1 mRNA in gastric fundus and duodenum.
In order to understand the relationship between the changes of regression-related molecules and other immunopathological changes in mucosa, we studied the changes of the transcription levels of inflammatory factors TNF-a and IFN-gamma after SIV/SHIV infection. Inflammatory factor TNF-alpha protein decreased significantly in jejunum and ileum, but decreased in small intestine and increased in large intestine as a whole; IFN-gamma protein did not change significantly, IL-6 protein increased significantly in ileum and cecum, and IL-17 protein increased significantly in rectum. It was found that FCGRT mRNA increased in intestinal mucosa, PIGR mRNA decreased in gastrointestinal tract and lymph nodes, BCL-6 mRNA decreased in small intestine and increased in large intestine after SIV/SHIV infection, while BCL-6 mRNA decreased in gastrointestinal mucosa (except rectum), spleen and lymph nodes after SHIV infection. The function of the disease may be affected.
CONCLUSIONS: In this study, the sequence of MAdCAM-1 cDNA in rhesus monkeys was obtained and found to be highly similar to that of human MAdCAM-1; 2) SIV/SHIV infection was found to affect the expression of MAdCAM-1 and receptor alpha 4 beta 7; 3) SIV/SHIV infection was found to affect the expression of multiple mucosal regression-related chemokines and receptors; 4) SIV/SHIV infection was found to affect the expression of multiple mucosal regression-related chemokines and receptors These results suggest that HIV-1 may affect mucosal and humoral immune function by influencing mucosal lymphocyte regression, and then influence mucosal cells and humoral immune function. It provides an important basis for understanding the pathological process of HIV/AIDS and finding new biological intervention targets.
【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】：2014
【分类号】：R512.91
，
本文编号：2179831
本文链接：https://www.wllwen.com/yixuelunwen/chuanranbingxuelunwen/2179831.html
最近更新
教材专著
