【作者】 李谦；

【导师】 施光峰；

【作者基本信息】 复旦大学 ， 内科学， 2009， 博士

【摘要】 慢性乙型病毒性肝炎（Chronic Hepatitis B，CHB）在我国是一种严重危害人类身体健康的传染性疾病，有近3500万的CHB患者。乙型肝炎病毒（Hepatitis B Virus，HBV）感染慢性化及其持续感染机制尚未完全阐明，故而临床上缺乏针对CHB的有效治疗方法。HBV感染慢性化机制复杂，HBV感染时机体免疫功能低下以致不能有效清除病毒是主要原因。功能性T细胞损伤是HBV持续感染的重要特征。T细胞被激活需要双刺激信号，均由抗原递呈细胞（antigen presenting cell，APC）提供。巨噬细胞（macrophage，MΦ）和树突状细胞（dendritic cell，DC）是专职的APC，DC还能够启动初始T细胞的免疫应答。单核细胞（Monocyte，Mo）则是巨噬细胞（macrophage，MΦ）和树突状细胞（dendritic cell，DC）的前体细胞，在淋巴细胞的极化和增殖中发挥重要的作用，同时能够决定人体与小鼠的初始及记忆性T细胞的反应格局。Mo不但在适应性免疫中扮演重要角色，在固有免疫中也发挥重要作用。Mo具有多种Toll样受体（Toll-like receptor，TLR），如表面的TLR2和TLR4等，胞内有TLR3等受体表达，能够感知外来抗原。细胞外信号可以被转换成胞内的生化事件，引起基因的转录激活和产物表达。单核细胞发挥作用除了可以通过细胞的直接作用或表面受体外，还可以分泌相关的细胞因子，如肿瘤坏死因子-α（Tumor necrosis factor-α，TNF-α）、白介素-6（Interleukine-6，IL-6）、IL-10和IL-12等。目前对Mo的研究正处于起步阶段；国内外都缺乏有关HBV慢性感染时Mo的系统性研究资料。因此，了解Mo的功能状态非常必要。本课题中，我们以Mo与HBV慢性感染时机体免疫功能低下的相关性为研究目的，从Mo对抗原感知，免疫应答和免疫调节三个阶段开展研究。第一部分慢性乙型肝炎患者外周血单核细胞亚群及TLR表达分析目的研究慢性乙型肝炎患者外周血单核细胞的比例、CD14^lowCD16⁺亚群及表面TLR2、TLR4的表达量，探讨单核细胞的CD14^lowCD16⁺亚群及TLR与HBV慢性感染之间的相关性。方法设立慢乙肝组（CHB组）和正常对照组（N组）。在慢乙肝组中筛选慢性乙型肝炎患者20例，未经任何抗病毒或免疫调节剂治疗，除外其他肝脏疾病及自身免疫性疾病；对照组中以12例健康者作对照。取外周抗凝血1ml，用流式细胞仪测定外周血Mos及CD14^lowCD16⁺亚群的比例和TLR2、TLR4的表达量。结果慢性乙型肝炎患者外周血中Mo的比例为8.53±2.39％，较之对照组的6.22±1.16％有明显增高（P＜0.05）。Mo亚群CD14lowCD16+在总Mo中所占的比例为14.56±7.31％，明显高于对照组的10.47±4.80％（P＜0.05）；慢乙肝组中表达TLR2的Mo比例为77.15±13.78％，平均荧光密度（mean flurescence intensity，MFI）为1358.12±612.49；对照组TLR2的Mo阳性率为77.02±8.31％，MFI为1322.15±251.02，两组比较无显著差异（p＞0.05）；慢乙肝组Mo的TLR4阳性率为76.88±10.27％，MFI 700.9±428.75；对照组的Mo TLR4阳性率是77.84±6.25％，MFI为614.0±235.64。两组之间比较TLR4的Mo阳性细胞表达率及MFI进行比较，显示差异无统计学意义（p＞0.05）。结论1．慢乙肝患者的外周血Mo比例及亚群CD14lowCD16+比例明显升高，提示单核细胞与HBV慢性感染有相关性；2．慢乙肝患者外周血Mo表面的TLR2和TLR4的表达并没有明显改变，提示Mo的TLR2和TLR4可能与HBV的慢性进展关联较小。第二部分慢性乙型肝炎患者外周血单核细胞的B7-H1及CD155转录水平的分析目的研究慢性乙型肝炎患者外周血单核细胞中，B7-H1和CD155的mRNA表达水平变化，探讨单核细胞在慢乙肝患者T细胞功能缺陷中的作用。方法设立CHB组和N组。在CHB组中筛选慢性乙型肝炎患者16例，未经任何抗病毒或免疫调节剂治疗，除外其他肝脏疾病及自身免疫性疾病；N组中以10例健康者作对照。从患者外周血中分离出单核细胞，以脂多糖（lipopolysaccharides，LPS）刺激，分别在刺激后0h、6h和24h收集细胞，制备总RNA，以Real-time PCR方法检测B7-H1和CD155 mRNA的表达水平。结果慢性乙型肝炎患者Mo在LPS刺激前，B7-H1 mRNA量为0.85±0.14，对照组在刺激前Mo的B7-H1 mRNA量为1.35±0.28，两组之间比较无显著统计学差异（p＞0.05）。LPS刺激6h后，慢乙肝组的B7-H1 mRNA量上升至1709±598.9，对照组则升至3179 1015，两组之间比较无明显差异（p＞0.05）；LPS刺激后24h，慢乙肝组的B7-H1 mRNA量是1271±492.3，而对照组的mRNA量是1716±535.84，差异无统计学意义（p＞0.05）。慢性乙型肝炎患者Mo在LPS刺激后6h，BT-H1的mRNA表达水平较刺激前有明显升高，差异具统计学意义（p＜0.05），但24h后未有继续升高；对照组的mRNA表达水平在LPS刺激后6h较刺激前有明显上升，差异具有显著性（p＜0.05），但是，刺激24h后B7-H1的mRNA表达水平有明显下降，但差异无统计学意义（p＞0.05）；慢性乙型肝炎患者Mo在LPS刺激前，CD155 mRNA量为1.52±0.49，对照组在刺激前，MoCD155的mRNA量为1.34±0.30，两组之间比较无明显统计学差异（p＞0.05）；LPS刺激后6h，慢乙肝组的CD155 mRNA量为1.97±0.41，而对照组为1.31±0.09，两组无明显差异（p＞0.05）；LPS刺激后24h，慢乙肝组的CD155mRNA量为3.56±0.14，对照组为6.30±1.30，比较无差异（p＞0.05）。慢性乙型肝炎患者Mo在LPS刺激后6h，CD155的mRNA表达水平较刺激前无明显变化，但是刺激24h后，CD155 mRNA的表达显著升高，差异具统计学意义（p＜0.05）；正常对照组中Mo的CD155 mRNA水平，在LPS刺激后6h较刺激前无明显变化（p＜0.05），但在刺激24h后，CD155的mRNA水平有明显升高，差异具有显著性（p＜0.05）。结论1．提示B7-H1和CD155与T细胞的功能受抑制无明显相关性；2．B7-H1和CD155为T细胞抑制性受体的配体，在LPS的刺激后表现为特征各异的时序性，可能是在不同的阶段发挥作用。第三部分慢乙肝患者外周血单核细胞LPS刺激后功能分析目的研究观察慢性乙型肝炎患者外周血单核细胞在LPS刺激后，分泌TNF-α、IL-6、IL-10和IL-12的情况，探讨单核细胞在HBV慢性感染时炎症反应能力及对T细胞功能的影响。方法设立CHB组和N组。CHB组中筛选慢性乙型肝炎患者16例，未经任何抗病毒或免疫调节剂治疗，排除其他肝脏疾病及自身免疫性疾病；N组中以10例健康者作对照。从外周血中分离出单核细胞，以LPS刺激，刺激后0h、6h及24h分别收集细胞培养上清液，用ELISA方法检测不同时间点TNF-α、IL-6、IL-10和IL-12的浓度。结果1．TNF-α的ELISA结果：1．1．两组间比较：LPS刺激前，CHB组TNF-α浓度为38.83±7.69 pg／ml，N组是35.40±7.72 pg／ml，两组间没有明显差异（p＞0.05）；刺激后6h，CHB组浓度升至5654±1350 pg／ml，N组为10320±51.05 pg／ml，N组显著高于CHB组（p＜0.05）；刺激后24h，CHB组TNF-α浓度为6384±175.9 pg／ml，N组是14770±780.0 pg／ml，明显高于CHB组，差异有统计学意义（p＜0.05）；1．2．不同时间点的比较：CHB组中，LPS刺激后6h的TNF-α浓度显著高于LPS刺激前（p＜0.05）；N组中，LPS刺激后6h的TNF-α浓度也显著高于刺激前（p＜0.05），刺激后24h的浓度较刺激后6h有显著升高（p＜0.05）。2．IL-6的ELISA结果：2．1．两组间比较：LPS刺激前，CHB组的浓度为1.804±0.60 pg／ml，N组是1.103±0.76 pg／ml，两组之间没有显著差异（p＞0.05）；刺激后6h，CHB组的IL-6浓度升至1133±68.91 pg／ml，N组为1069±26.93 pg／ml，两组差异不显著（p＞0.05）；刺激后24h，CHB组IL-6浓度为1084±11.81 pg／ml，N组的浓度是1600±16.79 pg／ml，对照组明显高于CHB组，差异有显著性（p＜0.05）；2．2．不同时间点的比较：慢乙肝组中，LPS刺激后6h的IL-6浓度明显高于LPS刺激前，差异有统计学意义（p＜0.05）；对照组中LPS刺激后6h的IL-6浓度明显高于刺激前，差异有统计学意义（p＜0.05），LPS刺激后24h的IL-6浓度较刺激后6h的浓度也有显著升高（p＜0.05）。3．IL-10的ELISA结果：3．1．两组间比较：LPS刺激前，CHB组的浓度为4.61±2.01 pg／ml，N组是13.14±4.39 pg／ml，两组之间没有明显统计学差异（p＞0.05）；刺激后6h，CHB组的IL-10浓度升至380.4±66.4 pg／ml，N组则为861.4±157.8 pg／ml，对照组明显高于慢乙肝组（p＜0.05）；刺激后24h，CHB组IL-10浓度为4241±914pg／ml，N组的浓度是2214±557.2 pg／ml，组间比较无差异（p＞0.05）；3．2．不同时间点的比较：慢乙肝组中，LPS刺激后6h浓度明显高于刺激前（p＜0.05），LPS刺激后24h的IL-10浓度明显高于刺激后6h（p＜0.05）；正常对照组中，LPS刺激后6h的IL-10浓度明显高于LPS刺激前，差异有统计学意义（p＜0.05），刺激后24h的浓度较刺激后6h的浓度也有显著升高（p＜0.05）。4．IL-12的ELISA结果：4．1．两组间比较：LPS刺激前，CHB组的浓度为8.87±1.70 pg／ml，N组是9.78±0.65 pg／ml，组间比较无差异（p＞0.05）；刺激后6h，CHB组的IL-12浓度升至21.40±2.96 pg／ml，N组则为21.70±13.23 pg／ml，组间比较无差异（p＞0.05）；刺激后24h，CHB组IL-12浓度为11.03±0.37 pg／ml，N组的浓度是16.80±1.56 pg／ml，组间比较无差异（p＞0.05）；4．2．不同时间点的比较：慢乙肝组中，LPS刺激后6h的IL-12浓度明显高于LPS刺激前，差异有统计学意义（p＜0.05）；对照组中，LPS刺激后6h的IL-10浓度明显高于刺激前，差异有统计学意义（p＜0.05）。结论1．LPS刺激后，单核细胞有TNF-α、IL-6、IL-10分泌，且呈时间依赖性；2．单核细胞受LPS诱导后有IL-12分泌，在刺激6h后达高峰；3．HBV慢性感染时，单核细胞功能有障碍，表现为炎性细胞因子分泌的减少。更多还原

【Abstract】 Chronic Hepatitis B (CHB) is a kind of severe contagious diseases.There are about 30 million CHB patients in our country. Up to now, thereare no effective therapies to treat this disease. The mechanism of HBVcontinuing infection is very complicated and not fully explained. One ofthe main reasons is that the function of immune system is impaired whenthe host is infected by HBV. One feature of HBV lasting infection is thefunctional impairment of T cell. The activation of T cell needs two kindsof signals, both of which are provided by APCs (antigen presenting cell).Both of macrophage and DC belong to APC, however only DC can activate na(i|¨)veT cell. Many studies have confirmed that monocyte is the precursor ofmacrophage and DC. Recent study suggest that monocyte can influence thepolarization and compassion of lymphocytes, and it can also shape thereaction of primary and memorial T cell of human and mouse.Monocyte is not only important in acquired immunity, but also in innateimmunity. Monocytes can recognize pathogen patterns with variousToll-like receptors(ThRs),such as TLR2 and TLR4 on the surface, and TLR3inside the cell. The activation of TLRs can induce cellular signaling inMo and therefore promote the production and secretion of related cytokines,such as TNF-α、IL-6、IL-10和IL-12, to regulate the immune response. Asa reason of that, the study of monocyte in CHB pathogenesis is veryimportant. However, the systemic research materials about monocyte andHBV chronic infection are still limited.The aim of my research is to investigate the relationship betweenmonocyte and the insufficiency of immunity in HBV chronic infection. Thestudy will focus on the three main functions of monocyte, which are antigenpresenting, inflammation reaction and immune regulation. PARTⅠPhenotype and subsets of peripheral monocyte fromchronic hepatitis B patientsObjectiveTo investigate the association phenotype and subsets of monocyteswith chronic hepatitis B.Method20 chronic hepatitis B patients were included in the CHB group, andthe twelve healthy were included in the control group. The expression ofTLR2 and TLR4 of monocyte was subjected to flow cytometric analysis. Andthe subsets of monocyte were analysed through flow cytometric analysis.ResultsThe percentage of blood monocyte in CHB group were 8.53±2.39%, Whichis higher than 6.22±1.16% of the N group; The percentage of CD14lowCD16+subsets were higher in CHB group (14.56±7.31%), than that of Ngroup(10.47±4.80%) (p＜0.05);TLR2: The percentage of TLR2 in CHB group were 77.15±13.78%,and MFIwas 1358.12±612.49; the percentage of monocyte TLR2 expression in Ngroup were 77.02±8.31%,and MFI was 1322.15±251.02; There is nosignificant difference between the two groups (p＞0.05).TLR4: The percentage of monocyte in CHB group were 76.88±10.27%, MFIwere 700.9±428.75; the percentage of monocyte in N group were 77.84±6.25%, MFI were 614.0±235.64; There were no difference between the twogroups (p＞0.05).Conclusion1. The positive correlation was found between elevated of percentagesof monocyte and HBV chronic infection. 2. There were no obvious relationship between the expression of TLR2、TLR4 of Mo and the dysfunction of T cell. PARTⅡThe expression of PD-H1 and CD155 of monocyte ofchronic hepatitis B patientsObjectiveTo investigate the association of of mRNA levels of B7-H1 and CD155with the function of T cell.MethodsThe research consisted of two groups: chronic hepatitis B group(CHBgroup) and normal group( N group). There were 16 CHB patients in the CHBgroup. They have not accepted any anti-virus or immune modulationtherapies in 6 months. And they have no other liver diseases or autoimmunediseases. The N group contains 10 healthy people. Monocytes were extractedfrom the peripheral blood, then these monocytes were stimulated by LPS.Mo were collected on 0h、6h and 24h post-stimulation. The level of BT-H1and CD155 mRNA were detected by Real-Time PCR.Results1. The level of BT-H1 mRNA:1.1. The mRNA level of B7-H1 were the same between CHB group(0.85±0.14)and N group(1.35±0.28) before the stimulation of LPS(p＞0.05): ThemRNA level of B7-H1 in normal group were 3179±1015, higher thanthat of CHB group(1709±598.9) (p＜0.05); The mRNA level of B7-H1in normal group were 1716±535.84 6 hours after stimulation, higherthan that of CHB group (1271±492.3) (p＜0.05);1.2. The mRNA level of B7-H1 were higer on 24 hours post-stimulation thanthat on 6 hours post-stimulation, the two groups are the same (p＜0.05).2. The level of CD155 mRNA:2.1. The mRNA level of CD155 were the same between CHB group(1.52±0.49)and N group(1.34±0.30) before the stimulation of LPS(p＞0.05); The mRNA level of B7-H1 in normal group were 1.31±0.09, higher than thatof CHB group(1.97±0.41) 6 hours after the LPS stimulation (p＜0.05); The mRNA level of B7-H1 in normal group were 6.30±1.30, higherthan that of CHB group(3.56±0.14) 24 hours after the LPS stimulation(p＜0.05);2.2. The mRNA level of CD155 were higher on 24 hours post-stimulation thanthat on 6 hours post-stimulation, the two groups are the same (p＞0.05).Conclusion1. There were no obvious relationship between the expression ofBT-H1、CD155 and the dysfunction of T cell;2. After the stimulation, the reaction of BT-H1 were earlier thanCD155 in bouth group; These two inhibitory receptors maybe take effectat different time. PARTⅢThe function of monocyte after stimulatin of LPS inchronic hepatitis B patientsObjectiveTo investigate the function of monocyte in HBV chronic infection andthe effect on T cell.MethodsThere were two groups: CHB group and N group. The CHB group contains16 patients. They have not received any anti-virus or immune modulationtherapies in 6 months. These patients have no other liver diseases andautoimmune diseases, 10 healthy people were included in N group. Themonocyte were extracted from blood. The protein level of TNF-α、IL-6、IL-10 and IL-12 were detected with ELISA.ResultsBefore the LPS stimulation, the concentration of TNF-αwas 38.83±7.69 pg/ml in CHB group, and 35.40±7.72 pg/ml in N group(p＞0.05);6 hours after the stimulation, the concentration of TNF-αwereelevated, and N group were 10320±51.05 pg/ml, higher than that of CHBgroup(5654±1350 pg/ml) (p＜0.05); 24 hours after the stimulation, theconcentration of TNF-αwere 14770±780.0 pg/ml, higher than that of CHBgroup(6384±175.9 pg/ml) (p＜0.05);Before the LPS stimulatin, the production of IL-6 in CHB group 1.804±0.60 pg/ml)was just the same as that of N group(1.103±0.76 pg/ml); Sixhours after the stimulation, the concentration of IL-6 higher in Ngroup(1069±26.93 pg/ml), higher than that of CHB group(1133±68.91pg/ml) (p＜0.05);Before the LPS stimulatin, the concentration of IL-10 were the samein both groups, N group were 13.14±4.39 pg/ml and CHB group were 4.61±2.01 pg/ml;After the stimulation, the concentration of IL-10 were higher on 6 hour, but there were the same between the two groups(p＞0.05).The N group were 861.4±157.8pg/ml, and CHB group were 380.4±66.4pg/ml. The level were 2214±557.2pg/ml in N group 24 hourspost-stimulation, the same as that of the CHB group(4241±914pg/ml) (p＞0.05).Before the LPS stimulatin, the production of IL-12 were the same inboth groups, N group were 9.78±0.65pg/ml and CHB group were 8.87±1.70pg/ml; After the stimulation, the production of IL-12 were higher on 6hour, but the same between the two groups(p＞0.05).The N group were 21.70±13.23pg/ml, and CHB group were 21.40±2.96pg/ml. The concentrationwere 16.80±1.56pg/ml in N group 24 hours post-stimulation, the same asthat of the CHB group(11.03±0.37pg/ml) (p＞0.05).Conclusion1. The monocyte can produce TNF-α、IL-6 and IL-10 after thestimulation of LPS, and the concentration were elevated during timecourse.2. The function of monocyte were impaired when HBV were chronicallyinfected, TNF-αand IL-6 were reduced after the stimulation of LPS whencompared with healthy group;3. There were no changes of IL-10 and IL-12 when HBV were chronicallyinfected, this suggest that the function of T cell were not influencedby monocyte through the production of cytokines IL-10 and IL-12.更多还原