慢性乙型肝炎患者外周血Th17和Treg细胞及其表面CXCR3的表达
发布时间：2018-09-17 19:17
【摘要】：背景和目的 中国约有9300万慢性HBV感染者，其中慢性乙型肝炎患者约2000万例。众所周知，乙型肝炎病毒引起不同程度的肝细胞损伤，并不是因为病毒直接杀伤肝细胞，而是由于病毒所诱导的免疫反应。其中，CD4+T淋巴细胞可以产生多种细胞因子，协助各特异性和非特异性效应细胞杀灭病毒，因而成为细胞和体液免疫反应的重要组成部分。而IL-17+辅助性和Foxp3+调节性T淋巴细胞是最近几年研究得比较多的对慢性乙型肝炎有重要作用的新型CD4+T淋巴细胞。已有研究显示：循环中IL-17+T(一般指Th17细胞)细胞大量聚集在慢性乙型肝炎患者的肝脏中，且表达频率随着肝炎程度的加重而增加；而Foxp3+T（一般指Treg细胞）细胞的功能与Th17细胞相反，它可抑制HBV抗原特异性T细胞反应，从而控制慢性乙型肝炎的进展；Th17与Treg细胞之间的平衡对慢性乙型肝炎的免疫稳态起着至关重要的作用。而CXCR3经典地表达于激活的人类CD4+T淋巴细胞表面，介导效应细胞趋化至外周炎症部位和淋巴结，且CXCR3的表达及其趋化因子配体IP-10也被报道与慢性乙型肝炎相关；但是，目前对CXCR3表达于Th17和Treg细胞表面的模式及其免疫调节作用却知之甚少。因此，本论文研究CXCR3在健康者以及不同程度慢性乙型肝炎患者中CD4+IL-17+T以及CD4+CD25+Foxp3+T淋巴细胞表面的表达频率，并初步探讨CXCR3+Th17和Treg细胞与肝损害之间的关系。 方法 收集不同程度慢性乙型肝炎患者以及健康者外周血2ml，排除其它原因引起的急慢性肝损伤，按照2000年中华医学会慢性乙型肝炎分级指南，分为健康对照组、轻中度慢性肝炎组、慢性重型肝炎组，提取出各组外周血中单个核淋巴细胞；利用流式细胞术，分别标记上Th17和Treg细胞抗原特异性标记抗体以及CXCR3单克隆抗体，后选用FACSCalibur流式细胞仪检测Th17、Treg细胞的比例以及其表面CXCR3的表达，FCS4软件进行分析；利用transwell技术分析IP-10对Th17和Treg细胞的趋化作用；SPSS18.0软件进行统计分析。 结果 与正常对照组相比，各肝炎组的外周血中Th17和Treg细胞比例均有所增加，尤其是慢性重型肝炎组；而肝炎组的Th17细胞中CXCR3+细胞比例与健康组差异不大，但是肝炎组的Treg细胞中CXCR3+细胞比例较健康组明显增加，，尤见于慢性重型肝炎组；按照肝脏ALT水平进行分级，CXCR3+Th17和CXCR3+Treg细胞在不同级别组有明显区别，肝脏损伤越严重（ALT越高），CXCR3+Th17细胞和CXCR3+Treg细胞比例就越高；趋化因子IP-10浓度越大，受趋化的CXCR3+Th17和Treg细胞就会越多。 结论 慢性乙型肝炎患者外周血Th17和Treg细胞比例随炎症损伤程度的增加而增加；且这些免疫细胞表面CXCR3的表达在不同状态下表达程度不一致，Th17细胞表面CXCR3的表达可以反映慢性肝炎时肝脏受损炎症程度；配体IP-10可与CXCR3相互作用，对Th17和Treg细胞发挥趋化作用。
[Abstract]:Background and objective there are about 93 million chronic HBV infections in China, including 20 million chronic hepatitis B patients. It is well known that hepatitis B virus causes varying degrees of hepatocyte damage, not because the virus directly kills the liver cells, but because of the immune response induced by the virus. Among them, CD4 T lymphocytes can produce a variety of cytokines to assist specific and non-specific effector cells to kill the virus, thus becoming an important part of cellular and humoral immune response. IL-17 adjuvant T lymphocytes and Foxp3 regulatory T lymphocytes are new type of CD4 T lymphocytes which have been studied in recent years and play an important role in chronic hepatitis B. It has been shown that circulating IL-17 T cells (generally referred to as Th17 cells) accumulate in the liver of patients with chronic hepatitis B, and the frequency of expression increases with the severity of hepatitis. The function of Foxp3 T cells is opposite to that of Th17 cells. It can inhibit the HBV antigen-specific T cell response and control the progression of chronic hepatitis B. The balance between Th17 and Treg cells plays an important role in the immune homeostasis of chronic hepatitis B. However, the expression of CXCR3 and its chemokine ligand IP-10 have been reported to be associated with chronic hepatitis B, and the expression of CXCR3 and its chemokine ligand IP-10 have been reported to be associated with chronic hepatitis B. Little is known about the expression of CXCR3 on the surface of Th17 and Treg cells and its immunomodulation. Therefore, we studied the expression frequency of CXCR3 on the surface of CD4 IL-17 T and CD4 CD25 Foxp3 T lymphocytes in healthy subjects and patients with chronic hepatitis B of different degrees, and explored the relationship between CXCR3 Th17 and Treg cells and liver damage. Methods Peripheral blood 2ml of patients with chronic hepatitis B and healthy subjects were collected to exclude acute and chronic liver injury caused by other causes. According to the classification guidelines of the Chinese Medical Association for chronic hepatitis B in 2000, they were divided into healthy control group. Mononuclear lymphocytes were extracted from peripheral blood of mild to moderate chronic hepatitis group and chronic severe hepatitis group. Th17 and Treg cell antigen specific labeling antibody and CXCR3 monoclonal antibody were labeled by flow cytometry. FACSCalibur flow cytometry was used to detect the proportion of Th17,Treg cells and the expression of CXCR3 on its surface. The transwell technique was used to analyze the chemotaxis of Th17 and Treg cells by using transwell technique. SPSS 18.0 software was used to analyze the chemotaxis of Th17 and Treg cells. Results compared with the normal control group, the percentage of Th17 and Treg cells in peripheral blood of the hepatitis group increased, especially in the chronic severe hepatitis group, but the proportion of CXCR3 cells in the Th17 cells in the hepatitis group was not different from that in the healthy group. However, the proportion of CXCR3 cells in Treg cells in hepatitis group was significantly higher than that in healthy group, especially in chronic severe hepatitis group. The proportion of CXCR3 Th17 cells and CXCR3 Treg cells increased with the increase of liver injury (ALT), and the more the concentration of chemokine IP-10, the more CXCR3 Th17 and Treg cells were chemotaxis. Conclusion the proportion of Th17 and Treg cells in peripheral blood of patients with chronic hepatitis B increased with the increase of inflammatory injury. The expression of CXCR3 on the surface of these immune cells was different in different states. The expression of CXCR3 on the surface of Th17 cells could reflect the degree of liver inflammation in chronic hepatitis, and the ligand IP-10 could interact with CXCR3 and play a chemotactic effect on Th17 and Treg cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62
本文编号：2246845
[Abstract]:Background and objective there are about 93 million chronic HBV infections in China, including 20 million chronic hepatitis B patients. It is well known that hepatitis B virus causes varying degrees of hepatocyte damage, not because the virus directly kills the liver cells, but because of the immune response induced by the virus. Among them, CD4 T lymphocytes can produce a variety of cytokines to assist specific and non-specific effector cells to kill the virus, thus becoming an important part of cellular and humoral immune response. IL-17 adjuvant T lymphocytes and Foxp3 regulatory T lymphocytes are new type of CD4 T lymphocytes which have been studied in recent years and play an important role in chronic hepatitis B. It has been shown that circulating IL-17 T cells (generally referred to as Th17 cells) accumulate in the liver of patients with chronic hepatitis B, and the frequency of expression increases with the severity of hepatitis. The function of Foxp3 T cells is opposite to that of Th17 cells. It can inhibit the HBV antigen-specific T cell response and control the progression of chronic hepatitis B. The balance between Th17 and Treg cells plays an important role in the immune homeostasis of chronic hepatitis B. However, the expression of CXCR3 and its chemokine ligand IP-10 have been reported to be associated with chronic hepatitis B, and the expression of CXCR3 and its chemokine ligand IP-10 have been reported to be associated with chronic hepatitis B. Little is known about the expression of CXCR3 on the surface of Th17 and Treg cells and its immunomodulation. Therefore, we studied the expression frequency of CXCR3 on the surface of CD4 IL-17 T and CD4 CD25 Foxp3 T lymphocytes in healthy subjects and patients with chronic hepatitis B of different degrees, and explored the relationship between CXCR3 Th17 and Treg cells and liver damage. Methods Peripheral blood 2ml of patients with chronic hepatitis B and healthy subjects were collected to exclude acute and chronic liver injury caused by other causes. According to the classification guidelines of the Chinese Medical Association for chronic hepatitis B in 2000, they were divided into healthy control group. Mononuclear lymphocytes were extracted from peripheral blood of mild to moderate chronic hepatitis group and chronic severe hepatitis group. Th17 and Treg cell antigen specific labeling antibody and CXCR3 monoclonal antibody were labeled by flow cytometry. FACSCalibur flow cytometry was used to detect the proportion of Th17,Treg cells and the expression of CXCR3 on its surface. The transwell technique was used to analyze the chemotaxis of Th17 and Treg cells by using transwell technique. SPSS 18.0 software was used to analyze the chemotaxis of Th17 and Treg cells. Results compared with the normal control group, the percentage of Th17 and Treg cells in peripheral blood of the hepatitis group increased, especially in the chronic severe hepatitis group, but the proportion of CXCR3 cells in the Th17 cells in the hepatitis group was not different from that in the healthy group. However, the proportion of CXCR3 cells in Treg cells in hepatitis group was significantly higher than that in healthy group, especially in chronic severe hepatitis group. The proportion of CXCR3 Th17 cells and CXCR3 Treg cells increased with the increase of liver injury (ALT), and the more the concentration of chemokine IP-10, the more CXCR3 Th17 and Treg cells were chemotaxis. Conclusion the proportion of Th17 and Treg cells in peripheral blood of patients with chronic hepatitis B increased with the increase of inflammatory injury. The expression of CXCR3 on the surface of these immune cells was different in different states. The expression of CXCR3 on the surface of Th17 cells could reflect the degree of liver inflammation in chronic hepatitis, and the ligand IP-10 could interact with CXCR3 and play a chemotactic effect on Th17 and Treg cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62
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