MDSCs与HBV慢性感染相关性的研究及其体外诱导机制的探索

发布时间：2018-05-21 01:26

  本文选题：慢性乙型肝炎 + 髓源性抑制性细胞(MDSCs)　； 参考：《山西医科大学》2014年硕士论文

【摘要】：研究背景和目的 乙型肝炎病毒(HBV)属于嗜肝非致细胞病变的DNA病毒,其持续感染可导致慢性肝炎,可进一步发展为肝硬化和肝癌。虽然对于HBV感染慢性化的潜在机制研究已有很多,但导致HBV持续性感染的患者体内抗病毒免疫应答的确切机制仍未完全阐明。本研究通过分析慢性乙型肝炎患者的外周血中髓源性抑制性细胞(MDSCs)的频率与健康人之间的差异、分析HBV成分与MDSCs频率的相关性,通过rHBsAg对MDSCs体外诱导作用及其可能机制的研究,旨在探讨MDSCs与HBV持续性感染的关系,为阐明MDSCs诱导HBV感染的免疫耐受机制提供实验依据,为HBV感染的免疫治疗提供新思路。 方法 (1)收集2013年6月至2013年9月期间在山西医科大学第一临床医院感染科住院治疗的慢性乙型肝炎患者肝素抗凝血标本,收集同期健康体检者血标本作为正常对照组。采用流式细胞术检测MDSCs频率,采用时间分辨免疫荧光分析法(TRFIA)检测乙型肝炎血清标志物,采用实时荧光定量PCR技术检测HBV DNA含量,并进行相应统计学分析。 (2)收集健康献血者的白细胞滤板,采用Ficoll密度梯度离心法分离PBMC进行细胞培养,然后用rHBsAg和PHA分别对其进行诱导刺激。诱导培养四天后采用多色FCM检测培养细胞中MDSCs比例,采用ELISA法检测细胞培养上清液中IFN-y含量,采用荧光定量PCR技术检测细胞中Arg-1、iNOS和p47phox的mRNA的转录水平。 结果 (1)慢性乙型肝炎组与健康对照组外周血中均含有一定比例的MDSCs,慢性乙型肝炎组MDSCs频率[2.10(1.23,3.30)%]显著高于正常对照组[0.54(0.43,0.76)%](P0.01)；HBsAg高含量组的MDSCs频率[3.30(2.40,5.30)%]显著高于低含量组[1.20(0.90,1.60)%](P0.001),且不同HBeAg含量间MDSCs频率的差异无统计学意义(P=0.097); MDSCs频率与WBC总数之间无相关性(P=0.170)。 (2) PBMC经体外培养后,rHBsAg刺激组MDSCs比例(8.93±1.23)%显著高于对照组(5.27±1.07)%(P=0.013); rHBsAg组上清液IFN-y含量[(34.30±7.98)]ng/ml与对照组[(33.05±7.60)ng/ml]之间的之间的差异无统计学差异(P=0.979),rHBsAg组Arg-1和p47phox的表达分别比对照组高1.81倍和5.26倍(P0.001),差别具有显著性意义,而iNOS的表达量组间比较差异无统计学意义(P=0.197)。 结论 (1)CHB患者外周血MDSCs频率较健康对照组显著增高,提示CHB患者对HBV特异应答低下与MDSCs的过度募集有关,而MDSCs介导的机体免疫耐受机制与乙型肝炎的迁延不愈有关。 (2) HBsAg含量高的患者MDSCs频率显著高于HBsAg含量低的患者,提示CHB患者外周血HBsAg可能在诱导MDSCs募集、生成过程中发挥重要作用。 (3) rHBsAg诱导MDSCs抑制机体清除病毒的免疫功能,可能是通过精氨酸酶途径以及ROS途径发挥作用。
[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 卤1.23%) was significantly higher than that in control group (5.27 卤1.07%), the content of IFN-y in supernatant of rHBsAg group [34.30 卤7.98)] ng/ml and control group [33.05 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

【参考文献】

相关期刊论文 前2条

1 李玲,顾长海,李欣;激活诱导细胞死亡在乙型肝炎发病机制中的意义[J];中华医学杂志;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

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本文编号：1917126