实验性自身免疫性葡萄膜炎模型中肝脏免疫系统紊乱的研究（英文）

葡萄膜炎是一种反复发作的炎症性疾病,可导致免疫系统功能障碍和多器官损伤.然而,葡萄膜炎是否导致肝功能损害尚不十分清楚.本文通过运用流式分析技术和激光共聚焦成像技术,研究了实验性自身免疫葡萄膜炎模型的肝脏病理和功能变化.结果显示肝损伤可出现在葡萄膜炎的炎症后期并与眼损伤程度相关.并且CD3~+CD4~+T细胞、CD3~-NK1.1~+DX5~-NK细胞、和CD11b~~+F4/80~-ly6c~+细胞在感染的眼睛和肝脏中增加.将CD3~+CD4~+T细胞回输给炎症的小鼠后,眼睛和肝脏的病理损伤加重.此外,在炎症的小鼠中可见血管扩张,大量淋巴细胞浸润到炎症的眼和肝脏的血管周围.总之,我们的研究结果提示,肝损伤可以发生在小鼠葡萄膜炎模型中,这种损伤可能与通过外周循环浸润到肝脏的CD3~+CD4~+T细胞有关.     

Toll样受体4在实验性自身免疫性葡萄膜炎发病中的作用

目的:研究常见感染菌菌壁成份脂多糖(LPS)在自身免疫性眼病-葡萄膜炎的致病作用.方法:用视网膜抗原IRBP和福氏完全佐剂免疫B10.RⅢ小鼠,诱发实验性自身免疫性葡萄膜炎(EAU),在免疫动物同时添加LPS.免疫19天后检查迟发型变态反应(DTH);21天处死小鼠,收集腹股沟淋巴结和髂动脉淋巴结细胞,检测抗原特异性T淋巴细胞增殖和炎症细胞因子的分泌.结果:LPS加重B10.RⅢ小鼠EAU的发病;增强DTH反应;增加抗原特异性T淋巴细胞增殖和IL-17、IFN-γ的分泌.结论:LPS能够促进EAU发病,从而证明病原微生物感染可能参与如葡萄膜炎等自身免疫性疾病的发生.     

急性肝损伤时肝脏内Pink1/Parkin的表达变化及意义

目的:观察C57小鼠急性肝损伤(Acute liver injury, ALI)中线粒体自噬相关蛋白Pink1/Parkin表达的变化及意义。方法:领取28只小鼠,随机分为对照组、ALI 1 d组、ALI 4 d组、ALI 7 d组。分别检测血清谷氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)的变化;肝组织病理变化用HE染色观察;Real Time quality PCR检测Pink1/Parkin在m RNA水平的表达变化;进一步利用Western Blot方法分别检测肝组织中Pink1/Parkin蛋白水平的表达变化。结果:ALI组1 d、4 d、7 d组与对照组相比,ALT、AST表达显著升高,具有统计学意义(P0.05);肝组织病理学检查ALI组1 d和4 d组中,释放出大量的炎性细胞,肝细胞出现大面积坏死,7 d组肝细胞坏死与1 d和4 d比较明显缓解;m RNA水平检测Pink1/Parkin在ALI 1 d(4.79/1.82倍)、4 d(1.74/4.17倍)和7 d(2.91/1.25倍)与对照组相比表达水平明显升高(P0.05);Pink1/Parkin在蛋白水平检测时ALI 1 d(2.03/1.82倍)、4 d(1.78/4.17倍)和7 d(1.25/1.25倍)时肝组织内表达也显著增加,差异具有统计学意义(P0.05)。结论:急性肝损伤状态下Pink1/Parkin在m RNA和蛋白水平表达升高,且其表达量随着肝脏的损伤缓解而表达水平降低,提示Pink1/Parkin可能对于治疗急性肝损伤具有重要意义。     

综述与专论: 肝脏免疫的活体显微光学成像研究进展

光学分子成像技术是在活体复杂的组织区域环境内细胞形态、运动与功能研究的最佳手段之一,极大地推进了免疫学的发展．肝脏是机体新陈代谢和解毒的重要器官,也被视为一个免疫器官．解析肝脏免疫基本特性和功能,对防治肝脏疾病以及全身性相关疾病具有重要意义．活体可视化研究肝脏区域生理或者病理状态下免疫应答,提供关键事件的多细胞参与及其彼此交互的时空动态信息,能极大地丰富对肝脏独特免疫反应的认知．本文将重点阐述目前活体肝脏成像的技术与方法以及光学显微成像技术,例如多光子激发显微成像与转盘共聚焦成像在肝脏免疫中的应用,并展望活体肝脏成像今后的发展方向和面临的机遇与挑战．     

耗竭性T细胞在肿瘤中的作用

耗竭性T细胞(exhausted T cells)是一群效应功能减弱,持续表达抑制性受体的T细胞,在肿瘤中表现为T细胞功能缺陷状态,主要特征为一系列抑制性受体表达增加及细胞因子分泌减少。耗竭性T细胞主要通过细胞表面的抑制性分子,细胞因子和免疫调节细胞类型改变等参与肿瘤免疫负调控,从而引起肿瘤免疫逃逸。而T细胞耗竭状态并非不可逆转,应用相应单克隆抗体靶向免疫调控点可以有效逆转耗竭性T细胞,恢复机体抗肿瘤免疫反应,提高肿瘤控制率。因此,通过逆转肿瘤患者体内的耗竭性T细胞可能是肿瘤免疫治疗的新途径之一。     

中暑致肝损伤机制的研究进展

中暑是常发生在夏季高温环境或大量运动时的急危重症,可导致包括肝脏在内的多器官功能损害。中暑的发生及发展过程经历了代偿期、急性反应期和失代偿期。近年来国内外关于中暑致肝损伤机制方面的研究表明,中暑致肝脏功能损伤可能与热的直接作用、肝细胞内线粒体功能障碍和级联放大的炎症反应有关,各环节相互促进,最终导致肝脏的损伤。而且,在肝窦内的级联放大炎症反应在中暑致肝损伤中可能起主要作用。因此,本文对近年来中暑的病理生理和中暑致肝损伤机制方面的研究及进展作一综述,为中暑致肝损伤的临床防治提供思路。     

芪参地黄颗粒对实验性自身免疫性重症肌无力大鼠B细胞介导的免疫机制研究

摘要 目的：探讨芪参地黄颗粒对实验性自身免疫性重症肌无力（EAMG）大鼠B细胞介导的免疫机制。方法：通过Rα97-116肽段和完全弗氏佐剂免疫,成功将30只Lewis大鼠构建EAMG模型,将EAMG大鼠随机分为模型组、芪参地黄颗粒低、中、高剂量组和阳性药组,每组6只。进一步观察大鼠体重及临床症状,检测血清中乙酰胆碱受体抗体（AChR-Ab）含量、脾脏组织CD19和CD27的蛋白表达、B淋巴细胞刺激因子（BAFF）、B细胞趋化因子CXC配体13（CXCL13）、C-X-C趋化因子受体5型（CXCR5） mRNA表达。结果：经给药治疗后芪参地黄颗粒低、中、高剂量组和阳性药组与模型组相比体重增加（P＜0.05）,临床症状评分均下降（P＜0.05）。经给药治疗后,与模型组相比,芪参地黄颗粒低、中、高剂量组血清中AChR-Ab含量均降低（P<0.05）,芪参地黄颗粒中、高剂量组脾脏组织CD27蛋白表达、CD19蛋白表达和BAFF mRNA表达降低（P＜0.05）,芪参地黄颗粒高剂量组脾脏组织CXCL13和CXCR5 mRNA表达降低（P＜0.05）,且芪参地黄颗粒中、高剂量组脾脏组织CD19蛋白表达较阳性药组下降（P＜0.05）。结论：芪参地黄颗粒通过降低EAMG大鼠CD19和CD27蛋白、BAFF、CXCL13和CXCR5 mRNA的表达,减少B细胞的分化增殖,抑制B细胞产生AChR-Ab,减少对乙酰胆碱受体的破坏,使EAMG大鼠体重增加,临床症状得到改善。     

哺乳动物PUF家族蛋白PUMILIO1的伴侣蛋白研究

目的:探索NIH3T3和DU145细胞中PUM1蛋白的伴侣蛋白。方法:收集NIH3T3和DU145细胞进行免疫沉淀实验,实验组和对照组分别使用抗PUM1抗体和抗Goat-Ig G抗体。通过银染实验观察实验组与对照组是否有差异性条带,通过质谱鉴定和Mascot软件对差异性条带所包含的蛋白进行分析,鉴定PUM1蛋白在细胞中发挥作用时的可能互动蛋白。结果:在NIH3T3和DU145细胞银染实验中,实验组凝胶泳道均在约37 KD处出现差异性条带。对NIH3T3细胞中差异性条带进行质谱分析,通过Mascot软件肽质量指纹谱搜索,将有真实差异的蛋白评分最低阈值设为65,P0.05,结果显示肽段评分最高的蛋白为NDFIP2蛋白。与NCBInr数据库比对,检测到针对NDFIP2的肽段的覆盖率为40%。结论:NDFIP2与哺乳动物PUM1蛋白在细胞内关联,可能是PUF家族蛋白的伴侣蛋白。     

原发性自身免疫性肝硬化的超声图征象及肝脏弹性的分析

摘要 目的：探讨与分析原发性自身免疫性肝硬化的超声图征象及肝脏弹性特征。方法：研究时间为2017年2月至2019年12月,选择原发性自身免疫性肝硬化86例作为病例组,同期选择正常志愿者86例作为对照组,所有入选者都给予常规超声与弹性成像,记录成像特征并判断相关性。结果：病例组的全血谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine transarninase,ALT)、直接胆红素(direct bilirubin,DBIL)、总胆红素(total bilirubin,TBIL)与碱性磷酸酶(Alkaline phosphatase,ALP值都显著高于对照组(P<0.05)。病例组的肝脏回声、边界、内回声、血流等超声特征与对照组对比差异都有统计学意义(P<0.05)。病例组肝脏组织剪切波速度(shear wave velocity,SWV)、肝硬度高于对照组,应变率比值(Strain rate, SR)值低于对照组,对比差异都有统计学意义(P<0.05)。病例组的肝脏弹性硬度分级与对照组对比差异有统计学意义(P<0.05)。在病例组中,Pearson分析显示肝脏弹性硬度分级与肝脏回声、边界、内回声、血流等存在相关性(P<0.05)。结论：常规超声及肝脏弹性可反映原发性自身免疫性肝硬化的临床特征,两者存在相关性,可提高该病的鉴别诊断水平。     

胃癌的过继性免疫治疗研究进展

胃癌是目前世界上发病率及致死率较高的恶性肿瘤之一,在东亚地区尤其显著。针对胃癌的治疗手段仍是传统的手术联合化疗、放疗为主,尽管靶向药物治疗提供了新的选择,但其对晚期胃癌的疗效仍然有限。胃癌的免疫治疗作为独特的治疗手段,在近十多年发展较为活跃,特别是过继性免疫治疗手段不断有创新。过继性免疫治疗主要依赖回输具有抗肿瘤活性的细胞,目前回输的细胞由具有非特异性抗肿瘤作用向具有特异性抗肿瘤作用演变,特别是嵌合性抗原T细胞治疗的出现,为进展期胃癌患者提供了有一种潜在的选择。本文对胃癌过继性免疫治疗中采用的不同免疫活性细胞的作用机制、临床应用等进行总结,并针对其不足提出利用基因工程技术增强治疗靶向性、降低免疫逃逸的研究方向。     

Liposome-Encapsulated Clodronate Retards the Development of Experimental Autoimmune Uveitis

Abstract

A study was undertaken to determine if the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (C1₂MDP; Clodronat), a treatment known to deplete monocytes, as well as liver and spleen macrophages, would reduce the number of macrophages in the retina of animals with experimental autoimmune uveitis (EAU) and decrease the severity of the disease. EAU was induced in Lewis rats by immunization with S-antigen (S-Ag). Monocytes and macrophages were depleted via an intravenous injection of Cl₂MDP encapsulated in liposomes. Control groups included rats that received no S-Ag (n= 18), S-Ag and no treatment (n=23), S-Ag and free drug (n = 20), or empty liposomes (n=14). Treated animals received injections of the Cl₂MDP-liposomes, free drug, or empty liposomes. Animals were sacrificed at 14, 21 and 28 days post-S-Ag administration. Intravenous, Cl₂MDP-liposomes produced a statistically significant reduction in the severity of the EAU when compared to controls at both days 14 and 21 following S-Ag injection. Immunohistochemical staining with the monoclonal antibody EDI demonstrated that the severity of the ocular inflammatory response correlated with the number of EDI-positive cells in the retina. Following the cessation of treatment, treated animals developed disease that was as severe at day 28 as that of untreated animals at day 21. These results confirm the importance of monocytes and macrophages in EAU by demonstrating the correlation between the presence of EDI-positive cells in the retina and the resultant damage to the retina. Although the dosing regimen employed here did not provide a cure, strategies designed to prevent the local recruitment and/or activation of mononuclear phagocytes may prove to be useful in the treatment of EAU.     

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.     

肝硬化患者的免疫功能障碍与血清壳多糖酶3样蛋白1表达的关系

摘要 目的：探究肝硬化患者的免疫功能障碍与血清壳多糖酶3样蛋白1（CHI3L1）表达关系。方法：招募2018年2月至2020年8月在我院就诊的160例肝硬化患者。通过Child-Pugh评分对肝病的严重程度进行分组,轻度组（n=73）和重度组（n=87）。在本院健康检查中心招募60例年龄相仿且无任何明显疾病或感染的人员作为对照组。并检测CHI3L1蛋白表达、CHI3L1浓度、MR浓度、CD3⁺T、CD25⁺T和CD69⁺T水平以及血浆细胞因子的浓度。结果：对照组较轻度和重度组AST、ALT浓度升高,且轻度组较重度组升高（P<0.05）。Cr血清浓度各组比较无差异（P>0.05）。轻度组中93.15 %患者为Child-Pugh A,重度组中全部为Child-Pugh B/A。重度组较轻度组肝硬化程度严重（P<0.05）。重度组和轻度组较对照组CHI3L1蛋白表达升高,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CHI3L1水平较对照组升高（P<0.05）,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CD163和甘露糖受体（MR）水平较对照组升高（P<0.05）,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CD3⁺T、CD25⁺T和CD69⁺T水平较对照组升高,重度组血清较轻度组升高（P<0.05）。重度组和轻度组白细胞介素（IL-1β）、IL-6、肿瘤坏死因子α（TNF-α）和干扰素（IFN-γ）浓度较对照组升高,重度组较轻度组升高（P<0.05）。Logistic回归分析,提示患者血清CHI3L1、MR、CD3⁺T、CD25⁺T、CD69⁺T与患者肝硬化程度相关（P<0.05）。结论：肝硬化患者中血清CHI3L1与肝硬化严重程度呈正相关,CHI3L1促进T细胞活化、增殖和炎性细胞因子的分泌,这导致加重了免疫介导的肝损伤和纤维化的发展。     

Brain and Spinal Cord Levels of Histamine in Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord homogenate emulsified with Mycobacterium tuberculosis-enriched complete Freund's adjuvant (CFA). Control rats were inoculated with CFA alone. Control and EAE rats were killed on days 7, 9, 11, and 13 postinoculation, and regional brain and spinal cord levels of histamine were determined. No regional differences in histamine content between control and EAE rats were seen on day 7 or 9 postinoculation. However, depending on the region, EAE rats exhibited significantly higher levels of histamine in their CNS on day 11 or 13 postinoculation or on both. Thus, regionally and temporally specific increases in brain and spinal cord levels of histamine develop concomitant with or just after the appearance (on day 10 postinoculation) of clinical signs of acute EAE, a finding suggesting that histamine may be involved in the development or expression of acute EAE in Lewis rats.     

Extrathymic Pathways of T-Cell Differentiation: A Primitive and Fundamental Immune System

In addition to an intrathymic pathway of T-cell differentiation, extrathymic pathways of T-cell differentiation have recently been demonstrated to occur in multiple sites in mice. Such sites include the sinusoids of the liver, the intraepithelial region of the intestine, and the omentum of the peritoneal cavity. Although these extrathymic pathways are minimal at a young age, they become predominant with aging. Extrathymically differentiated T cells display many properties distinct from those of regular T cells of thymic origin. For instance, they consist of a considerably large proportion of γδT cells as well as αβT cells, contain double-negative CD4^–CD8^– cells and self-reactive oligoclones, constitutively express the II-2 receptor β-chain, and have an αα homodimer of CD8 if they carry it. Cumulative evidence reveals that the extrathymic pathways comprise a primitive and fundamental immune system in the body and play a pivotal role in immune reactions under conditions of aging, bacterial infections, malignancies, autoimmune diseases, and pregnancy.     

综述与专论：免疫吸附治疗自身免疫疾病的研究进展

自身免疫疾病治疗的常规方法 (如免疫抑制剂和血浆置换等)缺乏足够的安全性和有效性,因此,寻找新的治疗方法十分必要。免疫吸附(immunoadsorption,IA)是一种通过选择性或非选择性去除自身致病抗体,从而实现对自身免疫疾病治疗的方法。本文介绍了免疫吸附在扩张型心肌病、特发性膜性肾病、系统性红斑狼疮、重症肌无力4种自身免疫疾病中的研究和临床应用,讨论了该治疗方法的有效性和安全性;同时指出,免疫吸附要在更多的疾病中实现临床应用,还需要在可信度、地域性、样本量等方面做更加深入的临床前试验。     

Age-Associated Increase of CD5+ B Cells in the Liver of Autoimmune (NZB × NZW) F1 Mice

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5⁺ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB × NZW) F₁ mice. An age-associated increase of CD5⁺ B cells was demonstrated in the liver of these mice. Although CD5⁺ B cells (i.e., CD5⁺IgM⁺ and CD5⁺B220⁺) constituted a minor population of hepatic mononuclear cells (MNC) (<5%) when mice were young (8 weeks), a large population of CD5⁺ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5⁺ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5⁺ B cells was the same as that of CD5⁺ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5⁺ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5⁺ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies. These results suggest that the liver might support the generation of the most primitive CD5⁺ B cells in these mice and that such generation increases as a function of age, probably resulting in the onset of autoimmune disease.     

CD8+T细胞在多发性硬化中的致病性作用

多发性硬化是T细胞介导的自身免疫性疾病。先前对它的研究大多集中在CD4 T细胞的致病和调节性作用上,但是,近几年来越来越多的证据表明CD8 T细胞也参与多发性硬化的病理损伤过程。 CD8 T细胞存在于MS病灶部位,髓鞘抗原特异性CD8 T细胞也从MS患者的血液和脑脊液中分离得到,CD8 T细胞通过直接杀伤或释放细胞因子和趋化因子等间接参与MS的病理过程。本文就近几年关于CD8 T细胞在多发性硬化中的致病性作用的研究进展予以介绍。     

肝脏免疫系统在酒精性肝损伤中的作用启示

酒精性肝病的致病因素是单一,但其发病机制复杂,目前尚不完全清楚。肝脏免疫系统被认为是独特的免疫系统,其作用越来越引起重视。肝内既有参与外周循环的淋巴细胞,也有长期定居于此的免疫细胞;加上肝脏解剖结构和血液循环的特殊性决定了肝脏独特的免疫微环境。研究肝脏免疫系统在酒精性肝病发病机制的作用,将有助于进一步阐明酒精性肝病发病机制,为酒精性肝病的预防和治疗提供新的靶点。