【作者】 李宁；

【导师】 施光峰； 蓝柯； 陈明泉；

【作者基本信息】 复旦大学 ， 内科学， 2010， 博士

【摘要】 DC是专职的抗原提呈细胞,是连接天然免疫与获得性免疫的桥梁,是平衡免疫应答与免疫耐受的关键。作为免疫系统的前沿哨兵,受到病原体或炎症信号的刺激后,DCs进行复杂的成熟过程,从而影响免疫应答的质与量。本研究旨在观察重型乙型肝炎(ACLF)患者外周血树突状细胞两个亚群(mDC和pDC)的频率,单核细胞衍生的树突状细胞(MoDC)表型及刺激同种异体T淋巴细胞增殖能力的变化。筛选重型乙型肝炎(ACLF)患者60例,慢性乙型病毒性肝炎(CHB)患者40例,并以20例健康者作对照,取入选研究对象的抗凝全血,应用流式细胞术检测外周血循环中mDC和pDC分别占外周血单个核细胞(PBMC)的比例。抗凝全血通过淋巴细胞分离液进行密度梯度法离心,分出PBMC,并进一步经免疫磁珠阳性选择的方法分离单核细胞,细胞因子白介素4和粒细胞巨噬细胞集落刺激因子体外诱导为MoDC,经poly(I:C)刺激后,用流式细胞仪检测MoDC的表型(HLA-DR、CD83、CD80、CD86),并通过同种混合淋巴细胞反应(MLR)检测MoDC刺激同种异体T淋巴细胞增殖的能力。1、健康对照组mDC和pDC占PBMC百分率分别为0.52%±0.04%和0.31%±0.02%；CHB组mDC和pDC占PBMC的百分率分别为0.53%±0.11%和0.2%±0.03%；ACLF组mDC和pDC占PBMC的百分率分别为0.18%±0.02%和0.15%±0.02%。ACLF组mDC占PBMC的百分率较CHB组及健康对照组均明显降低(P<0.001),但CHB组与健康对照组差异无统计学意义(P=0.927)。与健康对照组相比,ACLF组和CHB组pDC的百分率均显著降低(P<0.001),但ACLF组和CHB组之间差异无统计学意义(P=0.113)。2、经poly(I:C)刺激后健康对照组MoDC表面MHCⅡ类分子HLA-DR、成熟的标志CD83及协同刺激分子CD86和CD80平均荧光强度的表达分别为302.1±5.6、205.1±13.7、346.7±14.6、38.8±1.0；CHB组HLA-DR、CD83、CD86和CD80表达分别为272.2±3.38、173.0±6.4、315.5±7.93、30.6±0.57；ACLF组HLA-DR、CD83、CD86和CD80表达分别为223.0±5.5、148.2±6.1、219.1±7.1、25.0±0.6。CHB组MoDC四种表面标志物的表达均低于健康对照组,但高于ACLF组。3、MLR检测HBV感染者MoDC刺激T细胞增殖的能力低于健康对照组,重型乙型肝炎组降低尤为显著(P<0.001)。重型乙型肝炎患者外周血mDC和pDC占PBMC比率显著下降,MoDC表型缺失、成熟障碍以及功能低下,可能为重型乙型肝炎的发病机制之一。Toll样受体(TLR)是天然免疫系统中非常重要的一类模式识别受体,TLR3大量分布于树突状细胞,能识别病毒及其复制中间产物,TLR3触发后能促进核因子-κB (NF-κB)和干扰素调节因子3(IRF3)入核,继而分泌大量的促炎性细胞因子及干扰素β(IFN-β),可能与HBV感染的临床结局及预后相关。本研究观察慢性重型乙型肝炎患者外周血树突状细胞TLR3触发后信号转导通路的变化,探讨TLR3信号转导通路的改变与重型乙型肝炎疾病进展的相关性。筛选ACLF患者60例,CHB患者40例,并以20例健康者作对照,抽取入选研究对象的抗凝全血,通过淋巴细胞分离液进行密度梯度法离心和CD14+免疫磁珠阳性选择法分离单核细胞,细胞因子白介素4和粒细胞巨噬细胞集落刺激因子体外诱导为MoDC,经poly(I:C)刺激后,用流式细胞仪检测TLR3平均荧光强度(MFI),免疫印迹法检测TLR3蛋白水平,实时荧光定量PCR检测TLR3及TLR3信号通路上关键转录因子(核因子κB和干扰素调节因子3)的表达,用液相蛋白流式检测细胞培养上清中促炎性因子白细胞介素(IL)-6、IL-8、IL-10、IL-12p70、IL-1β和肿瘤坏死因子α(TNF-α)的分泌水平,酶联免疫吸附试验检测细胞培养上清中干扰素β(IFN-β)。1、相对于健康对照组,ACLF组MoDC上TLR3mRNA的表达下降57.7%,而CHB组下降41.5%,ACLF组较CHB组明显降低,三组之间具有显著性差异(P<0.001)。经免疫印迹法检测TLR3蛋白水平的表达趋势与转录水平一致,MoDC上TLR3的表达自健康对照组至CHB组至ACLF组依次减弱。MoDCs上TLR3的MFI在健康对照组明显高于两个疾病组(P<0.001)；而ACLF组TLR3 MFI低于CHB患者(P=0.006),差异具有统计学意义。2、健康对照组MoDC分泌IL-6、IL-8、IL-10、IL-12p70、TNF-α、IL-1β的水平分别为：(711.64±61.64)ng/L、(5893.53±377.53)ng/L、(51.66±9.72)ng/L(39±0.89)ng/L、(7370.01±597.66)ng/L、(40.55±4.72)ng/L；CHB组MoDC分泌IL-6、TNF-α、IL-12p70、IL-10、IL-1β和IL-8的水平分别为：(1081.64±82.5)ng/L、(4181.66±413.35)ng/L、(59.44±10.37)ng/L、(36.44±2.66)ng/L、(12627.67±924.33)ng/L、(27.75±2.49)ng/L；ACLF组MoDC分泌IL-6、TNF-α、IL-12p70、IL-10、IL-1β和IL-8的水平分别为：(1551±161)ng/L、(2959.86±415.58)ng/L、(30.62±2.97)ng/L、(29±1.03)ng/L、(19263.61±1110.58)ng/L、(25.12±1.05)ng/L；ACLF组MoDCs分泌IL-6和TNF-α显著高于CHB和健康对照组,IL-12p70、IL-10、IL-1β和IL-8分泌异常。3、MoDCs分泌IFN-β水平在各组之间的差异与TLR3表达趋势相同,健康对照组为(156.10±6.98) pg/ml,CHB组为(133.95±5.45)pg/ml, ACLF组为(74.77±3.1) pg/ml。CHB组MoDCs分泌IFN-β水平低于健康对照组(P=0.019)；ACLF组MoDCs分泌IFN-β水平低于CHB组(P<0.001),更低于健康对照组(P<0.001)。4、针对ACLF患者进一步分析,ACLF中存活组TLR3/IFN-β显著高于死亡组(P<0.001),而TLR3/IFN-β与HBeAg状态无显著相关性。ACLF组患者NF-κB mRNA显著高于CHB和健康对照组,而IRF3 mRNA低于CHB和NC组。与重型肝炎相关的临床参数相关性分析发现TLR3/IFN-β的分泌水平与凝血酶原活动度呈负相关,与总胆红素成正相关,而与谷丙转氨酶和病毒载量无显著相关性。重型乙型肝炎患者TLR3信号转导通路受损,促炎性细胞因子分泌异常,Ⅰ型干扰素分泌降低可能是导致肝细胞严重免疫损伤及HBV持续感染的原因之更多还原

【Abstract】 Dendritic cells (DCs) are potent antigen-presenting cells (APCs), which bridge innate and adaptive immune responses and shape the balance between induction of immunity and tolerance. As sentinels, DCs alert the immune system, and determine the quantity and quality of the emerging immune response. Persistence of hepatitis B virus (HBV) infection is associated with reduced anti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers, however, its role in hepatitis B virus (HBV)-related chronic-on-acute liver failure is still largely unknown. To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B related acute-on-chronic liver failure, we assayed the circulating precursor DC subsets (including mDC and pDC) and the ability of MoDCs in patients at various stages of HBV infection.Peripheral blood was collected from hepatitis B related acute-on-chronic liver failure patients (ACLF, n=60) and chronic hepatitis B (CHB, n=40) as well as normal controls (NCs, n=20). Circulating mDC and pDC frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis. Purified monocytes were isolated by combination of Histopaque-1.077 and CD 14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I:C. Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR)of MoDCs were detected in patients with hepatitis B 1. The peripheral mDC and pDC frequency in normal controls were 0.52%±0.04% and 0.31%±0.02%, respectively. In addition, the peripheral mDC and pDC frequency in patients with CHB were 0.53%±0.11% and 0.2%±0.03%, respectively. Those of ACLF were 0.18%±0.02% and 0.15%±0.02%. The number of circulating mDC decreased only in patients with hepatitis B related ACLF compared with that in normal controls. However, pDC numbers decreased in both CHB and ACLF patients. We observed a further decrease the pDC numbers in ACLF compared to CHB patients, however, but this difference did not achieve P< 0.05 statistical significance.2. After in vitro challenge with poly(I:C), the expression of NC-DC surface molecular such as MHC classⅡmolecules (HLA-DR), and the mature marker CD83, as well as costimulatory molecules CD86, CD80 were 302.1±5.6,205.1±13.7,346.7±14.6, and 38.8±1.0, respectively. But both groups showed low expressions of above surface molecular (272.2±3.38,173.0±6.4,315.5±7.93, and 30.6±0.57 in CHB group; 223.0±5.5,148.2±6.1,219.1±7.1, and 25.0±0.6 in ACLF group). MoDC from ACLF patients showed lower expression of costimulatory molecules CD80, CD86 and the mature marker CD83, as well as MHCⅡmolecule (HLA-DR) compared to CHB and NC group.3. Interestingly, MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared with those in CHB patients and NCs.Our results suggest that patients with hepatitis B related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC, as well as decreased number of circulating mDC and pDC, which may be partially related to HBV disease progression in these patients. Toll-like receptors (TLRs) are a class of proteins that play key roles in innate immunity through recognition of microbial components. TLR3 is expressed abundantly in dendritic cells, and is responsible for recognizing viral pathogens and inducing proinflammtory cytokine and interferon beta (IFN-β) production. Although TLR3 has been reported to be involved in several diseases caused by viral infections, its role in hepatitis B virus (HBV)-induced hepatitis is still largely unknown. This study aims to investigate the expression of TLR3 signaling in monocyte derived dendritic cells (MoDC) from ACLF, to assess the contribution of TLR3 in ACLF.Peripheral blood was collected from ACLF patients (n=60) and chronic hepatitis B (CHB, n=40) as well as normal controls (n=20). Purified monocytes were isolated by combination of Histopaque-1.077 and CD 14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by TLR3 ligand (poly I:C). TLR3 pathway genes of extracted RNA were detected using real-time PCR. The level of TLR3 protein was measured by flow cytometric analysis and western blotting. Supernatant fluids from the cultures were analyzed for levels of 6 different pro-inflammatory cytokines, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-a by flow cytometric, and interferonβwas measured by ELISA.1. We found that expression of TLR3 mRNA was decreased significantly in monocyte-derived dendritic cells (MoDCs) from patients with CHB (41.5%) or ACLF (57.7%), compared with NCs. Particularly in ACLF patients, the TLR3 expression was further decreased under that in CHB patients (P<0.001). Data from western blotting and flow cytometry analysis further confirm the above observation at the protein level. In addition, the MFI of TLR3 was lower in HBV infection patients than NC group (P< 0.001), we observed a further decrease in TLR3 MFI in ACLF compared to CHB patients (P=0.006).2. Interestingly, ACLF-MoDC produced imbalanced relative levels of pro-inflammatory cytokines, particularly IL-6 and TNF-α, were higher than CHB patients and controls. While, aberrant amounts were found of IL-12p70, IL-10, IL-1β, and IL-8 on MoDCs in ACLF.3. The secretion of IFN-βfrom DC of control group, patients with CHB and ACLF were (156.10±6.98) pg/ml, (133.95±5.45)pg/ml, and (74.77±3.1) pg/ml, respectively. The secretion of IFN-βwas mirrored on the expression of TLR3. The secretion of IFN-βwas lower in patients with CHB and ACLF than NC group (P< 0.001). We observed a further decrease in IFN-P in ACLF compared to CHB patients (P< 0.001).4. Compared with surviving patients, TLR3 and IFN-βexpression was significantly lower in non-surviving ACLF patients, which strongly indicated a correlation between TLR3 signaling impairment in MoDCs and disease severity in ACLF patients. Further linear correlation analysis demonstrated significant correlations between expression of TLR3 signaling components (TLR3 and IFN-β) and disease severity markers (prothrombin activity and total bilirubin) for individual ACLF patients.The results suggest impaired TLR3 pathway signaling in MoDC from patients with ACLF, resulting in secretion of selective pro-inflammatory cytokines and IFN-βintegral to the inflammatory response that may be critical in the pathogenesis of ACLF.更多还原