实验性自身免疫性肝炎的研究

本文通过 S-100肝抗原多次免疫同种 C57BL/6小鼠后,成功地建立了实验性自身免疫性肝炎的动物模型,并在此基础上进一步探讨了过继输注致敏脾淋巴细胞给正常同种小鼠后自身免疫性肝炎的发生情况.结果表明,导致过继输注后自身免疫性肝炎主要为 T 淋巴细胞,揭示了 T 淋巴细胞在自身免疫性肝炎发病中起着重要作用.另外,本文还就免疫抑制剂环孢素 A 对实验性自身免疫性肝炎的防治作用进行了探讨.组织学检查结果表明预防和治疗用药时均能减轻肝脏病理变化和/或减少自身免疫性肝炎的发生率.为临床治疗自身免疫性肝炎提供了实验室依据.     

FXR通过调节促甲状腺素胚胎因子减轻Con A诱导的自身免疫性肝炎病变

 目的：观察法尼酯衍生物X受体（farnesoid X receptor,FXR)-促甲状腺素胚胎因子（thyrotropin embryonic factor,TEF）通路在自身免疫性肝炎模型小鼠肝损害中的作用,探讨FXR-TEF通路改善自身免疫性肝炎的部分可能机制。方法：检测FXR在伴刀豆球蛋白A (concanavalin A, Con A) 诱导的肝炎（Con A-induced hepatitis, CIH）小鼠肝脏的表达;检测FXR激活对TEF表达的影响;观察C57BL/6小鼠和鹅去氧胆酸（chenodeoxycholic acid,CDCA）激活FXR的CIH小鼠肝脏病理、肝脏酶学及炎症因子变化。结果：FXR在CIH小鼠中低表达;CDCA激活FXR的C57BL/6小鼠TEF表达上调;FXR被激活的CIH小鼠的肝损害较轻,FXR激活可减轻肝脏炎症因子释放。结论：CDCA激活FXR能减轻CIH引起的肝功能损害和炎症反应。FXR激活使TEF上调。FXR可能是自身免疫性肝炎的保护因素,其保护作用可能是通过TEF来实现的。激活FXR可能成为治疗自身免疫性肝炎的一个途径。     

人CYP2D6诱导小鼠自身免疫性肝炎动物模型的建立

目的研究模拟抗原人细胞色素P450家族2亚家族D成员6(CYP2D6)和遗传背景差异的小鼠(C57BL/6和BALB/c)对自身免疫性肝炎(AIH)动物模型建立的影响。方法 C57BL/6和BALB/c小鼠尾静脉注射腺病毒及质粒p CYP2D6,用ELISA检测血清抗CYP2D6抗体和抗肝蛋白自身抗体水平; HE染色检测肝脏AIH炎症程度,天狼猩红染色检测肝纤维化的程度;微孔板法检测血清丙氨酸转氨酶(ALT)的水平。结果腺病毒促进人CYP2D6诱导C57BL/6小鼠产生抗肝蛋白自身抗体、并引起AIH和肝纤维化,而在BALB/c小鼠中,仅产生针对人CYP2D6的抗体,而无抗肝蛋白自身抗体、不产生AIH和肝纤维化。结论在腺病毒存在下,模拟抗原人CYP2D6在肝脏中的持续表达可以诱导C57BL/6小鼠产生AIH,但是无法诱导BALB/c小鼠产生AIH,不同品系小鼠的遗传背景可能影响着AIH的发生发展。     

联合阻断CD28/B7和ICOS对同种异体C57BL/6小鼠皮肤移植的影响

目的探讨CD28/B7共刺激通路阻断剂和ICOS/ICOSL共刺激通路阻断剂对同种异体小鼠皮肤移植的影响。方法实验动物随机分为4组,每组6只,供体、受体均为C57BL/6。CTLA4Ig处理组;ICOSIg处理组;CTLA4Ig+ICOSIg处理组;空白对照组。将C57BL/6小鼠背部全层皮肤移植于同种小鼠中段背部。前3组分别以150mg/kg的抗体经腹腔注射,空白对照组只注射同等量的生理盐水,注射时间为皮肤移植后0、2、4d,术后观察皮肤存活情况。术后6d分别处死各组受体鼠和供体鼠,取受体脾脏淋巴细胞与供体脾脏淋巴细胞作混合淋巴细胞反应(MLR),用MTT法测定细胞活力和细胞增殖反应。术后7d切取皮肤移植物作组织学分析。结果与对照组相比,各阻断剂单独治疗组能明显延长移植物的存活时间(<0.05),各阻断剂治疗组淋巴细胞对供体淋巴细胞产生特异性低反应(<0.05),能有效抑制淋巴细胞对同种抗原的免疫应答。组织学检查发现,各抗体治疗组移植皮肤结构基本正常。结论 CTLA4Ig、ICOSIg可以抑制免疫应答,诱导移植皮肤发生免疫耐受,延长存活时间。     

趋化因子CCL2在实验性自身免疫性脑脊髓炎中的作用

目的:探讨趋化因子CCL2在实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)中的作用及其可能机制。方法:选取CCL2~(-/-)敲除小鼠和C57BL/6小鼠,利用髓鞘少突细胞糖蛋白(Myelin oligodendrocyte glycoprotein,MOG33-35)建立EAE小鼠模型,观察小鼠发病情况,并根据Konot神经功能评分标准对其进行评分;待模型组小鼠发病评分普遍为4~5分时,收集CCL2~(-/-)、C57BL/6模型组和C57BL/6空白组小鼠(n=10)脊髓组织进行HE染色观察和免疫组化检测,分析小鼠脊髓组织的病理变化和CD4~+、CD8~+T淋巴细胞数量改变。结果:CCL2~(-/-)、C57BL/6模型组小鼠发病率分别为0%、100%;其中,1只CCL2~(-/-)模型组小鼠在免疫后第21天出现轻微拖尾随后恢复正常。免疫后第21天时,CCL2~(-/-)模型组小鼠的神经功能评分明显低于C57BL/6模型组(P0.05)。HE染色结果显示,C57BL/6模型小鼠的脊髓组织中脱髓鞘现象明显,其炎性细胞数目显著高于CCL2~(-/-)组(P0.05);同时,免疫组化结果表明,CCL2~(-/-)小鼠脊髓组织中CD4~+T淋巴细胞、CD8~+T淋巴细胞均分别低于C57BL/6小鼠对照组(P0.05),尤其CD8~+细胞量改变存在显著差异(P0.05)。结论:趋化因子CCL2可能通过抑制T淋巴细胞的浸润抑制EAE的发生发展。     

Resolvin D1预处理通过NF-κB及焦亡影响失血性休克肝损伤

目的 探讨Resolvin D1预处理对失血性休克小鼠肝脏损伤的保护作用及可能机制.方法 30只雄性C57BL/6小鼠,随机分成Sham组、HS/R组及Resolvin D1组,每组10只.血生化检测各组小鼠肝脏功能;HE染色观察肝脏形态学变化;ELISA检测炎症因子水平;Western Blot检测焦亡相关蛋白NLR...     

荷EL4肿瘤小鼠模型的建立及美法仑抑瘤作用免疫机制的探讨

目的:建立荷小鼠淋巴瘤EL4的野生型C57BL/6小鼠及其裸鼠模型,探讨美法仑(melphalan)抑瘤作用的免疫机制。方法:给正常野生型C57BL/6小鼠皮下接种小鼠淋巴瘤EL4细胞,建立荷EL4肿瘤的小鼠模型。于野生型C57BL/6小鼠皮下接种瘤细胞后12d,经腹腔给荷瘤小鼠单次注射不同剂量的美法仑,找出美法仑可发挥最大的抑瘤作用,并能致使肿瘤消退、不再复发的最小使用剂量。然后再给野生型C57BL/6小鼠及其裸鼠(遗传背景相同)皮下同时接种小鼠淋巴瘤EL4细胞建立两种荷瘤小鼠模型。同样于接种瘤细胞后12d,经腹腔给两种荷瘤小鼠模型均注射可使野生型C57BL/6小鼠肿瘤消退、不再复发的最低剂量的美法仑,以正常野生型C57BL/6小鼠为对照,观察在T淋巴细胞缺陷的裸鼠体内美法仑的抑瘤作用。结果:注射7.5mg/kg美法仑治疗后,免疫功能正常的野生型C57BL/6荷瘤小鼠的肿瘤消退;而荷瘤C57BL/6裸鼠的肿瘤仍继续生长。结论:单一剂量的美法仑对荷淋巴瘤EL4小鼠具有明显的治疗作用,其作用的发挥需要T淋巴细胞的参与,可能与T细胞的杀伤作用有关。     

盐酸小檗碱通过抑制TNF-α/caspase-8/caspase-3信号通路减轻急性低氧暴露引起的肝损伤

目的 研究盐酸小檗碱(BBR)对急性低氧暴露后肝脏的保护作用及相关机制.方法 C57BL/6小鼠分为常氧组、低氧暴露组、低氧暴露联合BBR组.实验第7天处死小鼠,取肝脏组织.称量小鼠及肝组织的质量,利用HE染色观察肝组织病变情况,利用实时定量PCR检测肝组织白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF...     

化疗治愈荷瘤小鼠模型的建立及氟尿嘧啶抑瘤作用的免疫机制

目的:建立荷瘤野生型C57BL/6小鼠及C57BL/6裸鼠模型,探讨5-氟尿嘧啶(5-FU)抑瘤作用的免疫机制。方法:给正常野生型C57BL/6小鼠皮下接种小鼠淋巴瘤EL4细胞,建立荷EL4肿瘤的小鼠模型。接种瘤细胞后12d,荷瘤小鼠腹腔单次注射不同剂量的5-FU,找出5-FU可发挥最大的抑瘤作用、并能致使荷瘤小鼠肿瘤消退不再复发的最小使用剂量。遗传背景相同的野生型C57BL/6小鼠及C57BL/6裸鼠同时皮下接种小鼠淋巴瘤EL4细胞,建立两种荷瘤小鼠模型。接种瘤细胞后12d,两种荷瘤小鼠腹腔内同时注射可使野生型C57BL/6荷瘤小鼠肿瘤消退不再复发5-FU的最低剂量,以正常野生型C57BL/6小鼠为对照,观察5-FU对T淋巴细胞缺陷裸鼠的抑瘤作用。结果:以75mg/kg5-FU治疗1周后,两种荷瘤小鼠的肿瘤以相同的速率缩小直至消失。但在5-FU治疗2周后,T淋巴细胞缺陷的C57BL/6裸鼠肿瘤复发,而免疫功能正常的野生型C57BL/6小鼠肿瘤治愈。结论:单一剂量的5-FU对荷淋巴瘤EL4小鼠具有明显的近期治疗效果,5-FU抑瘤作用的发挥不需要T细胞参与;但5-FU抗肿瘤作用的远期疗效与机体T淋巴细胞功能密切相关。     

MOG诱导小鼠EAE模型的建立及相关免疫指标的变化

目的用髓鞘少突胶质细胞糖蛋白35-55(MOG)多肽免疫C57BL/6小鼠建立实验性自身免疫35-55性脑脊髓炎(EAE)模型,探讨EAE模型发病期与相关免疫指标的关系。方法用MOG抗原免疫C57BL/6雌35-553性小鼠,观察其临床症状和中枢神经系统的病理改变;应用H方法测定EAE小鼠脾脏T淋巴细胞活化增殖程度;++应用流式细胞术检测中枢及外周脾脏淋巴细胞中CD4 CD25调节性T细胞;荧光定量RT-PCR检测IL-17A、Foxp3、IFNγ的表达水平。结果成功建立EAE模型。与对照组相比,EAE组中可见小鼠中枢神经系统有淋巴细+++胞浸润,脾脏T淋巴细胞增殖随MOG多肽浓度增加而增强,CD4 CD25调节性T细胞占CD4 T细胞的比例明显降35-55低(P0.01),IL-17和IFNγm RNA表达量显著升高(P0.05),Foxp3 m RNA表达量显著降低(P0.05)。结论 MOG多肽成功诱导了C57BL/6小鼠EAE模型,Th17与Treg细胞共同参与调控EAE的病理过程。35-55     

CTLA4Ig修饰树突状细胞对实验动物淋巴细胞增殖及胞毒效应的影响

目的：探讨CTLA4Ig修饰的DC对实验动物免疫功能的影响。方法：将经CTLA4Ig基因修饰或未修饰Dc腹腔注射C57BL／6致敏小鼠，以致敏或未致敏C57BL／6单个核细胞作为反应细胞，以未修饰DC细胞及修饰DC为刺激细胞，共培养6天，采用MTT比色法检测细胞增殖，乳酸脱氢酶法测定细胞毒活性。结果：CTLA4Ig融合蛋白对未修饰DC致敏或未致敏小鼠的同种细胞刺激的增殖反应有明显的抑制作用。CTLA4Ig修饰DC诱导不同组小鼠淋巴细胞增殖反应均明显降低，CTLA4Ig融合蛋白对CTL细胞毒活性有显著抑制作用。CTLA4Ig修饰DC对不同组小鼠CTL细胞毒活性均具有抵抗作用，未修饰DC细胞对未致敏小鼠以及未修饰DC对致敏小鼠CTL细胞毒活性敏感。结论：稳定表达CTLA4Ig融合蛋白的DC诱导显著降低同种小鼠淋巴细胞的增殖反应和对CTL细胞毒活性的抵抗。     

Effect of anti-CD134L mAb and CTLA4Ig on ConA-induced proliferation, Th cytokine secretion, and anti-dsDNA antibody production in spleen cells from lupus-prone BXSB mice

We sought to evaluate the effects of combined downregulation of CD134 and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) on the autoimmune process of lupus. Concanavalin A (ConA)-induced proliferation, T helper cell cytokine secretion, and anti-double stranded DNA (dsDNA) antibody production were measured in cultures of splenic lymphocytes derived from lupus-prone BXSB mice. Splenocytes from six prednisone-treated and six untreated male lupus-prone BXSB mice, as well as from six syngeneically normal C57BL/6 male mice, were stimulated with ConA. BXSB splenocytes from untreated mice were exposed to anti-CD134L mAb, CTLA4 linked to the Fc portion of IgG1 (CTLA4Ig), or both. The magnitude of splenocyte proliferation and the levels of IFN-gamma, IL-6, and anti-dsDNA antibody were: (1) significantly higher in cultures of ConA-stimulated control and other cells than in unstimulated cells, (2) similar in cultures of normal and BXSB cells treated with anti-CD134 and CTLA4Ig or prednisone and (3) significantly reduced in cultures of ConA-stimulated and unstimulated cells treated with anti-CD134L and CTLA4Ig or prednisone compared with cells treated with CD134L or CTLA4Ig alone. Like corticosteroids, anti-CD134L mAb or CTLA4Ig can inhibit T- and B-cell activation by blocking the CD134-CD134L or CD28/CTLA4-B7 co-stimulatory pathway. The combined immune intervention described herein may prove useful for the treatment of autoimmune diseases such as systemic lupus erythematosus.     

Type 2 autoimmune hepatitis murine model: the influence of genetic background in disease development

Genetic predisposition is recognized as an important factor for the development of autoimmune hepatitis (AIH). To assess the potential contribution of MHC and non-MHC genes, type 2 AIH was reproduced in three mice strains, taking advantage of their different genetic makeup with regard to MHC and non-MHC genes. Mice (C57BL/6, 129/Sv and BALB/c) were DNA vaccinated with a pCMV-CTLA4-CYP2D6-FTCD plasmid coding for the extracellular region of CTLA-4 and for the antigenic region of the CYP2D6 and FTCD, and with pCMV-IL12. ALT and total IgG levels, liver histology, FACS analysis and liver T-cell cytotoxicity assays were monitored up to 8 months post-injection. C57BL/6 mice showed elevated serum ALT levels, autoantibodies, antigen-specific cytotoxic T-cells and lobular and periportal inflammatory infiltrate. The 129/Sv mice showed slightly elevated ALT levels, sparse liver lobular infiltrate and cytotoxic T-cells. The BALB/c mice showed no liver inflammation. All mice had elevated total serum IgG levels. This murine model of type 2 AIH shows that MHC and non-MHC genes contribute to the susceptibility to autoimmune hepatitis. The understanding of the genetic determinants implicated in AIH development will be a major advance in the study of its pathogenesis and could lead to a better diagnostic approach and preventive strategies.     

Immunohistopathological reactions for liver-specific membrane lipo-protein in experimental autoimmune hepatitis

Antibody to the hepatocyte membrane protein, was induced in inbred strain C57BL/6 and C3H mice by immunisation with 100,000 g supernatant of syngeneic liver homogenate in CFA. Three weekly intraperitoneal injection of 200 ul of liver homogenate with CFA for continuous 4 weeks gave the best possible result. Histopathological changes were characterised mainly by perivascular inflammatory infiltrates and hepatocyte necrosis which mimicked human autoimmune hepatitis. In one of the immunological parameters, antibody to hepatocyte membrane protein (LSP) has been demonstrate by ouchterlony method in the test serum of those animals, who had received weekly doses of liver antigen. Thus in experimental autoimmune liver disease, semi-purified syngeneic liver fluid (S-100) leads to hepatic destruction and to an inflammatory process with several features in common with human chronic aggressive hepatitis. The presence of antibody against syngeneic liver antigen (S-100) in the test sera emphasizes that hepatocyte membrane protein does have an important role in liver tissue pathogenesis and disease process in experimental model. In this study we tried to prove that hepatocyte membrane protein may act as a target antigen in developing experimental autoimmune hepatitis.     

Study of cellular immunity in experimental autoimmune hepatitis in mice.

This study was undertaken to produce experimental autoimmune hepatitis in mice, and to examine the role of liver specific lipoprotein (LSP), if any, and of cellular immunity in such a model. After immunization of three strains of mice (C57BL/6, C3H/He and BALB/c) with syngeneic crude liver proteins, most prominent liver changes histologically mimicking human hepatitis were produced in the liver of C57BL/6 (B6) mice. Antigenic and immunogenic activity of LSP in the crude liver proteins was decreased by the treatment of freezing and thawing, and the recovery of the antigenic activity seemed to correlate with the susceptibility of immunized mice to the induction of liver damage. Autoantibody against LSP was demonstrated in the serum of immunized B6 mice, but not in the sera of other strains after immunization. It was also found that EDTA contained in the buffer used for purification of LSP distinctly suppressed lymphocyte activity in vivo and in vitro. With the use of EDTA free LSP, it was shown that spleen cells of immunized B6 mice (especially of T cell enriched fraction) had a high reactivity studied by lymphocyte transformation test. Further examination showed that EDTA free LSP could induce mild liver lesions and lymphocyte reactivity against LSP, although neither histological change nor lymphocyte reactivity was found in the liver of B6 mice immunized with EDTA containing LSP.     

Exploration the significance of Tfh and related molecules on C57BL/6 mice model of experimental autoimmune hepatitis

BackgroundThis study aimed at establishing the autoimmune hepatitis (AIH) model of C57BL/6 mice, and examining the expression and significance of T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells, effector B cells and other indicators in this experimental autoimmune hepatitis (EAH) model.MethodsC57BL/6 mice in experimental group were administered by intraperitoneal injection after fully emulsified on 1st day and 7th day with 0.5 mL of 0.5–2.0 g/L S-100 and an equal volume of Buddha Complete Adjuvant (CFA). The levels of serum alanine transaminase (ALT), aspartate aminotransferase (AST), and interleukin-21 (IL-21) were tested by the corresponding kit. Tfr, Tfh and B cell subsets were quantified by flow cytometry. Histological pathology was completed by pathological section experiments.ResultsIn comparison with that in the healthy controls (HC), significantly increased numbers of serum ALT and AST, Tfr, IL-21 was observed. The hepatic lobules and hepatocyte cords were severely disorganized.ConclusionThe results of liver pathological changes and serum index changes were similar to the chronic and progressive pathogenesis and pathology of AIH patients, providing a detailed pathological basis for the basic research and clinical experiments of AIH.     

Unique B cell responses in B cell-dependent and B cell-independent EAE

Previous studies characterized B cell-dependent and B cell-independent models of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. To further characterize the B cell response generated in these two models, the serum antibody response and the B cell surface immunoglobulin (Ig) repertoire were analyzed following immunization of wild-type C57BL/6 mice with either recombinant myelin oligodendrocyte glycoprotein (MOG; B cell-dependent EAE) or the encephalitogenic MOG(35-55) peptide (B cell-independent EAE). Plasma ELISA revealed responses to unique linear epitopes of MOG following immunization with recombinant MOG that were absent in MOG(35-55)-immunized animals. B cell repertoire analysis by RT-PCR identified a unique response restricted to 7183 Ig heavy chain variable gene family in mice immunized with recombinant MOG that was not observed in MOG(35-55)-immunized mice. These insights could aid in the identification of the relevant B cell populations important to the pathogenesis of B cell-dependent EAE and in the mechanisms by which these B cell populations contribute to disease.     

乙型肝炎病毒转基因小鼠C57-TgN(adr2.0)SMMU品系的免疫病理研究

目的: 研究乙型肝炎病毒 (HBV)转基因小鼠C57 TgN(adr2. 0型)SMMU品系的免疫病理学特征, 并与临床人慢性乙肝相比较。方法: 以 20只SPF级肝脏有明显病变的HBV转基因小鼠为研究对象, 用间接免疫荧光法通过流式细胞仪, 分别检测转基因小鼠及正常C57BL/6小鼠外周血淋巴细胞表面CD3、CD4和CD8表达的水平, 同时取其肝组织和5例本院病理科存档确诊为慢性中度乙型肝炎患者的肝组织石蜡标本, 用EnVision免疫组化染色法检查肝组织内T细胞亚群的分布。结果: 转基因小鼠外周血淋巴细胞表面CD3、CD4和CD8表达的水平低于正常对照组小鼠; 肝组织内浸润的单个核细胞多数为CD3 CD4 细胞, 未发现CD57 、CD8 细胞。慢性乙型肝炎患者的肝组织内浸润的单个核细胞主要为CD3 CD4 或CD3 CD8 细胞和少量CD57 细胞。结论: C57 TgN(adr2. 0型 )SMMU的HBV转基因小鼠外周血及肝组织中CD的表达与人慢性乙肝患者的外周血及肝组织有明显不同。     

Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis

Cytotoxic T lymphocyte antigen 4 (CTLA‐4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co‐stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)‐β receptor II dominant negative (dnTGF‐βRII) transgene to address the potential therapeutic efficacy of CTLA‐4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA‐4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA‐4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF‐βRII mice, CTLA‐4 Ig treatment has short‐term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA‐4 Ig will be critical to determining whether such efforts can be applied to human PBC.