【摘要】：背景和目的 中國約有9300萬慢性HBV感染者，其中慢性乙型肝炎患者約2000萬例。眾所周知，乙型肝炎病毒引起不同程度的肝細(xì)胞損傷，并不是因?yàn)椴《局苯託渭?xì)胞，而是由于病毒所誘導(dǎo)的免疫反應(yīng)。其中，CD4+T淋巴細(xì)胞可以產(chǎn)生多種細(xì)胞因子，協(xié)助各特異性和非特異性效應(yīng)細(xì)胞殺滅病毒，因而成為細(xì)胞和體液免疫反應(yīng)的重要組成部分。而IL-17+輔助性和Foxp3+調(diào)節(jié)性T淋巴細(xì)胞是最近幾年研究得比較多的對(duì)慢性乙型肝炎有重要作用的新型CD4+T淋巴細(xì)胞。已有研究顯示：循環(huán)中IL-17+T(一般指Th17細(xì)胞)細(xì)胞大量聚集在慢性乙型肝炎患者的肝臟中，且表達(dá)頻率隨著肝炎程度的加重而增加；而Foxp3+T（一般指Treg細(xì)胞）細(xì)胞的功能與Th17細(xì)胞相反，它可抑制HBV抗原特異性T細(xì)胞反應(yīng)，從而控制慢性乙型肝炎的進(jìn)展；Th17與Treg細(xì)胞之間的平衡對(duì)慢性乙型肝炎的免疫穩(wěn)態(tài)起著至關(guān)重要的作用。而CXCR3經(jīng)典地表達(dá)于激活的人類CD4+T淋巴細(xì)胞表面，介導(dǎo)效應(yīng)細(xì)胞趨化至外周炎癥部位和淋巴結(jié)，且CXCR3的表達(dá)及其趨化因子配體IP-10也被報(bào)道與慢性乙型肝炎相關(guān)；但是，目前對(duì)CXCR3表達(dá)于Th17和Treg細(xì)胞表面的模式及其免疫調(diào)節(jié)作用卻知之甚少。因此，本論文研究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淋巴細(xì)胞表面的表達(dá)頻率，并初步探討CXCR3+Th17和Treg細(xì)胞與肝損害之間的關(guān)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損傷，按照2000年中華醫(yī)學(xué)會(huì)慢性乙型肝炎分級(jí)指南，分為健康對(duì)照組、輕中度慢性肝炎組、慢性重型肝炎組，提取出各組外周血中單個(gè)核淋巴細(xì)胞；利用流式細(xì)胞術(shù)，分別標(biāo)記上Th17和Treg細(xì)胞抗原特異性標(biāo)記抗體以及CXCR3單克隆抗體，后選用FACSCalibur流式細(xì)胞儀檢測(cè)Th17、Treg細(xì)胞的比例以及其表面CXCR3的表達(dá)，F(xiàn)CS4軟件進(jìn)行分析；利用transwell技術(shù)分析IP-10對(duì)Th17和Treg細(xì)胞的趨化作用；SPSS18.0軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果 與正常對(duì)照組相比，各肝炎組的外周血中Th17和Treg細(xì)胞比例均有所增加，尤其是慢性重型肝炎組；而肝炎組的Th17細(xì)胞中CXCR3+細(xì)胞比例與健康組差異不大，但是肝炎組的Treg細(xì)胞中CXCR3+細(xì)胞比例較健康組明顯增加，，尤見于慢性重型肝炎組；按照肝臟ALT水平進(jìn)行分級(jí)，CXCR3+Th17和CXCR3+Treg細(xì)胞在不同級(jí)別組有明顯區(qū)別，肝臟損傷越嚴(yán)重（ALT越高），CXCR3+Th17細(xì)胞和CXCR3+Treg細(xì)胞比例就越高；趨化因子IP-10濃度越大，受趨化的CXCR3+Th17和Treg細(xì)胞就會(huì)越多。 結(jié)論 慢性乙型肝炎患者外周血Th17和Treg細(xì)胞比例隨炎癥損傷程度的增加而增加；且這些免疫細(xì)胞表面CXCR3的表達(dá)在不同狀態(tài)下表達(dá)程度不一致，Th17細(xì)胞表面CXCR3的表達(dá)可以反映慢性肝炎時(shí)肝臟受損炎癥程度；配體IP-10可與CXCR3相互作用，對(duì)Th17和Treg細(xì)胞發(fā)揮趨化作用。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.62

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