本文選題：慢性乙型肝炎 + 髓源性抑制性細(xì)胞(MDSCs)　； 參考：《山西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景和目的 乙型肝炎病毒(HBV)屬于嗜肝非致細(xì)胞病變的DNA病毒,其持續(xù)感染可導(dǎo)致慢性肝炎,可進(jìn)一步發(fā)展為肝硬化和肝癌。雖然對于HBV感染慢性化的潛在機制研究已有很多,但導(dǎo)致HBV持續(xù)性感染的患者體內(nèi)抗病毒免疫應(yīng)答的確切機制仍未完全闡明。本研究通過分析慢性乙型肝炎患者的外周血中髓源性抑制性細(xì)胞(MDSCs)的頻率與健康人之間的差異、分析HBV成分與MDSCs頻率的相關(guān)性,通過rHBsAg對MDSCs體外誘導(dǎo)作用及其可能機制的研究,旨在探討MDSCs與HBV持續(xù)性感染的關(guān)系,為闡明MDSCs誘導(dǎo)HBV感染的免疫耐受機制提供實驗依據(jù),為HBV感染的免疫治療提供新思路。 方法 (1)收集2013年6月至2013年9月期間在山西醫(yī)科大學(xué)第一臨床醫(yī)院感染科住院治療的慢性乙型肝炎患者肝素抗凝血標(biāo)本,收集同期健康體檢者血標(biāo)本作為正常對照組。采用流式細(xì)胞術(shù)檢測MDSCs頻率,采用時間分辨免疫熒光分析法(TRFIA)檢測乙型肝炎血清標(biāo)志物,采用實時熒光定量PCR技術(shù)檢測HBV DNA含量,并進(jìn)行相應(yīng)統(tǒng)計學(xué)分析。 (2)收集健康獻(xiàn)血者的白細(xì)胞濾板,采用Ficoll密度梯度離心法分離PBMC進(jìn)行細(xì)胞培養(yǎng),然后用rHBsAg和PHA分別對其進(jìn)行誘導(dǎo)刺激。誘導(dǎo)培養(yǎng)四天后采用多色FCM檢測培養(yǎng)細(xì)胞中MDSCs比例,采用ELISA法檢測細(xì)胞培養(yǎng)上清液中IFN-y含量,采用熒光定量PCR技術(shù)檢測細(xì)胞中Arg-1、iNOS和p47phox的mRNA的轉(zhuǎn)錄水平。 結(jié)果 (1)慢性乙型肝炎組與健康對照組外周血中均含有一定比例的MDSCs,慢性乙型肝炎組MDSCs頻率[2.10(1.23,3.30)%]顯著高于正常對照組[0.54(0.43,0.76)%](P0.01)；HBsAg高含量組的MDSCs頻率[3.30(2.40,5.30)%]顯著高于低含量組[1.20(0.90,1.60)%](P0.001),且不同HBeAg含量間MDSCs頻率的差異無統(tǒng)計學(xué)意義(P=0.097); MDSCs頻率與WBC總數(shù)之間無相關(guān)性(P=0.170)。 (2) PBMC經(jīng)體外培養(yǎng)后,rHBsAg刺激組MDSCs比例(8.93±1.23)%顯著高于對照組(5.27±1.07)%(P=0.013); rHBsAg組上清液IFN-y含量[(34.30±7.98)]ng/ml與對照組[(33.05±7.60)ng/ml]之間的之間的差異無統(tǒng)計學(xué)差異(P=0.979),rHBsAg組Arg-1和p47phox的表達(dá)分別比對照組高1.81倍和5.26倍(P0.001),差別具有顯著性意義,而iNOS的表達(dá)量組間比較差異無統(tǒng)計學(xué)意義(P=0.197)。 結(jié)論 (1)CHB患者外周血MDSCs頻率較健康對照組顯著增高,提示CHB患者對HBV特異應(yīng)答低下與MDSCs的過度募集有關(guān),而MDSCs介導(dǎo)的機體免疫耐受機制與乙型肝炎的遷延不愈有關(guān)。 (2) HBsAg含量高的患者M(jìn)DSCs頻率顯著高于HBsAg含量低的患者,提示CHB患者外周血HBsAg可能在誘導(dǎo)MDSCs募集、生成過程中發(fā)揮重要作用。 (3) rHBsAg誘導(dǎo)MDSCs抑制機體清除病毒的免疫功能,可能是通過精氨酸酶途徑以及ROS途徑發(fā)揮作用。
[Abstract]:Background and purpose of the study Hepatitis B virus (HBV) is a non-cytopathic DNA virus. Its persistent infection can lead to chronic hepatitis and further develop into liver cirrhosis and liver cancer. Although much research has been done on the underlying mechanism of chronic HBV infection, the exact mechanism of antiviral immune response in patients with persistent HBV infection has not been fully elucidated. In this study, the frequency of myelogenous inhibitory cells (MDSCs) in peripheral blood of patients with chronic hepatitis B (CHB) was analyzed, and the correlation between the HBV components and the frequency of MDSCs was analyzed. The in vitro induction of MDSCs by rHBsAg and its possible mechanism were studied. The aim of this study was to explore the relationship between MDSCs and persistent infection of HBV, to provide experimental basis for elucidating the mechanism of immune tolerance induced by MDSCs in HBV infection, and to provide a new idea for immunotherapy of HBV infection. Method From June 2013 to September 2013, heparin anticoagulant blood samples were collected from patients with chronic hepatitis B who were hospitalized in the first Hospital infection Department of Shanxi Medical University. The frequency of MDSCs was detected by flow cytometry, the serum markers of hepatitis B were detected by time-resolved immunofluorescence assay (TRFIA), and the content of HBV DNA was detected by real-time fluorescence quantitative PCR. (2) the leukocyte filter plate of healthy blood donors was collected, PBMC was isolated by Ficoll density gradient centrifugation, and then stimulated by rHBsAg and PHA respectively. After induced culture for four days, polychromatic FCM was used to detect the proportion of MDSCs in cultured cells, ELISA method was used to detect the content of IFN-y in the supernatant of cell culture, and fluorescence quantitative PCR technique was used to detect the mRNA transcription level of Arg-1iNOS and p47phox in cultured cells. Result The frequency of MDSCs in chronic hepatitis B group was significantly higher than that in normal control group [0.54 0.43 ~ 0.76%] MDSCs frequency in high content group [3.302.405.30%] was significantly higher than that in low content group [1.200.900.60%] P0.001a, and different HBeAg levels were significantly higher than those in normal control group [0.540.43] 0.76%, respectively. The frequency of MDSCs in chronic hepatitis B group was significantly higher than that in low content group [1.20 ~ 0.90 ~ 1.60%] P _ (0.001), and the frequency of MDSCs was significantly higher than that in normal control group [0.54] 0.43 ~ 0.43 ~ 0.76%]. There was no significant difference in MDSCs frequency between the two groups, but there was no correlation between the MDSCs frequency and the total number of WBC. (2) the ratio of MDSCs in PBMC stimulated group (8.93 鹵1.23%) was significantly higher than that in control group (5.27 鹵1.07%), the content of IFN-y in supernatant of rHBsAg group [34.30 鹵7.98)] ng/ml and control group [33.05 鹵7.60)ng/ml] had no significant difference. The expression of Arg-1 and p47phox in P0.979rHBsAg group was 1.81 times higher than that in control group. 5.26 times P 0.001, the difference was significant, However, there was no significant difference in the expression of iNOS between the two groups. Conclusion The frequency of MDSCs in peripheral blood of CHB patients was significantly higher than that of healthy controls, suggesting that the low response to HBV in CHB patients was related to the overrecruitment of MDSCs, while the mechanism of immune tolerance mediated by MDSCs was related to the persistence of hepatitis B. (2) the frequency of MDSCs in patients with high HBsAg content was significantly higher than that in patients with low HBsAg content, suggesting that peripheral blood HBsAg in CHB patients might play an important role in inducing MDSCs recruitment and production. RHBsAg induces MDSCs to inhibit the immune function of virus clearance, which may be mediated by arginase pathway and ROS pathway.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李玲,顧長海,李欣;激活誘導(dǎo)細(xì)胞死亡在乙型肝炎發(fā)病機制中的意義[J];中華醫(yī)學(xué)雜志;2003年13期

2 Yi-Min Zhang;Yi-Da Yang;Hong-Yu Jia;Lin-Yan Zeng;Wei Yu;Ning Zhou;Lan-Juan Li;;HBsAg levels in HBeAg-positive chronic hepatitis B patients with different immune conditions[J];World Journal of Gastroenterology;2014年15期

，

本文編號：1917126