ANGPTL8敲除减轻DEN诱导的小鼠急性肝损伤

目的 探讨血管生成素样蛋白8(ANGPTL8)在N-二乙基亚硝胺（DEN）诱导小鼠急性肝损伤中的作用及机制。方法 选取雄性野生型（WT）和ANGPTL8敲除（ANGPTL8 KO）C57BL/6J小鼠，腹腔注射DEN(50 mg/kg)诱导急性肝损伤模型（15日龄各组32只，8周龄各组14只）。利用PCR和免疫荧光检测肝组织和肝原代细胞中ANGPTL8的表达变化；试剂盒检测血清中ALT、AST含量；HE染色观察肝脏组织病理学变化；流式细胞术和共聚焦显微镜分析肝原代细胞活性氧（ROS）累积差异；ELISA检测血液中炎症因子IL-6、IL-1β含量；TUNEL染色检测肝细胞凋亡水平。结果 DEN诱导3 d显著上调肝脏中ANGPTL8表达；与WT小鼠相比，ANGPTL8 KO可显著下调DEN诱导小鼠血清中ALT和AST、IL-6和IL-1β水平；ANGPTL8KO小鼠肝小叶紊乱程度、炎症细胞浸润、肝细胞淤血、空泡样变和坏死程度较轻；ANGPTL8 KO小鼠显著改善DEN诱导的肝细胞ROS累积和凋亡。结论 DEN上调肝脏中ANGPTL8表达，ANGPTL8进一步促进肝脏急性炎症期ROS累积、炎...     

Lnk基因敲除对葡聚糖硫酸钠诱导小鼠结肠炎的影响

目的比较Lnk基因敲除小鼠与野生型小鼠在葡聚糖硫酸钠(DSS)诱导结肠炎后的表现。方法将周龄相近的C57BL/6小鼠分为野生型组(WT)、野生型小鼠结肠炎组(WT+DSS)、Lnk敲除鼠组(KO)和Lnk敲除鼠结肠炎组(KO+DSS)。WT和KO小鼠正常饮水,WT+DSS及KO+DSS组小鼠自由饮用2. 5%DSS水溶液。实验进行7 d,每日观察小鼠体质量变化、粪便软硬度及肠道出血情况,评估疾病活动指数(DAI)。7 d后取外周血,采用流式细胞技术检测WT小鼠和KO小鼠外周血中调节性B细胞(Breg)频率。处死小鼠,取肠观察外形、颜色,测量肠的长度。结肠组织制作为组织学切片,进行HE染色,显微镜下观察组织学改变。结果 WT和KO组小鼠排便正常,KO+DSS组小鼠与WT+DSS组小鼠较早出现腹泻及排血便情况。KO+DSS组小鼠的体质量在实验周期内较其他3组有显著降低,KO+DSS组DAI随时间显著增加。KO组Breg细胞频率显著低于WT组。KO+DSS组结肠明显变短,HE组织切片镜检糜烂出血、淤血,多病灶浅溃疡,炎性细胞浸润黏膜及黏膜下层并累及肌层,提示炎症反应加重。结论 Lnk缺失可加重DSS诱导的小鼠结肠炎表现,可能与Lnk KO小鼠体内Breg细胞频率降低、对炎症反应的负调控作用减弱有关。     

白细胞介素-6/糖蛋白130/转录激活因子3通路在百草枯诱导小鼠急性肺损伤中的作用机制

目的基于肺特异性白细胞介素-6(IL-6)敲除小鼠研究IL-6/糖蛋白130(gp130)/转录激活因子3(STAT3)通路在百草枯(PQ)诱导急性肺损伤(ALI)中的作用。方法野生型C57BL/6J小鼠分为IL-6野生型(IL-6 WT)组、IL-6 WT+PQ组,采用Sftpc(肺表面活性蛋白C基因)-Cre⁺小鼠与IL-6^FLOX/FLOX小鼠交配的方式得到肺特异性IL-6敲除小鼠并分为IL-6 KO组、IL-6 KO+PQ组,单次腹腔注射PQ诱导ALI,比较四组小鼠肺组织病理改变、肺泡动脉氧分压差(PA-aO₂)、肺组织湿/干质量比值(W/D)、IL-6/gp130/STAT3通路、核转录因子-κB(NF-κB) p65、肿瘤坏死因子-α(TNF-α)及白细胞介素-1β(IL-1β)的差异。结果与IL-6 WT组比较,IL-6 WT+PQ组小鼠肺组织出现了典型的ALI病理改变,肺组织胞浆蛋白中IL-6、gp130、p-STAT3、TNF-α、IL-1β表达水平及胞核蛋白中NF-κB p65的表达水平、PA-aO₂、W/D明显增加(P <0.05);与IL-6 WT+PQ组比较,IL-6 KO+PQ组小鼠肺组织病理改变明显改善,肺组织胞浆蛋白中IL-6、gp130、p-STAT3、TNF-α、IL-1β的表达水平及胞核蛋白中NF-κB p65的表达水平、PA-aO₂、W/D明显降低(P <0.05)。结论 IL-6/gp130/STAT3通路激活与PQ诱导ALI有关。     

垂体肿瘤转化基因1通过调控肠上皮细胞焦亡在结肠炎症中的作用

目的 明确垂体肿瘤转化基因1(PTTG1)通过调控肠上皮细胞的焦亡水平在结肠炎症中的作用机制。方法 PTTG1野生型（WT）小鼠和PTTG1基因敲除（KO）小鼠各10只,随机分为4组,每组5只,分别为PTTG1 WT对照组（control组）和实验组（3%DSS组）,PTTG1 KO对照组和实验组。实验组小鼠自由饮用3%葡聚糖硫酸钠（DSS）6 d诱导急性实验性结肠炎,对照组给予无菌双蒸水。观察并记录各组小鼠的疾病活动指数（DAI）。收集小鼠结肠组织,用免疫组织化学和蛋白免疫印迹法检测焦亡相关蛋白NLRP3、ASC、GSDMD的表达水平。在人源结肠上皮细胞系（HcoEpic）中,通过shRNA沉默PTTG1的表达,TNF-α刺激细胞以诱导细胞炎症模型,检测GSDMD的表达水平。结果 DSS诱导的结肠炎小鼠结肠黏膜组织中PTTG1表达减少（P <0.01）,PTTG1敲除加重小鼠结肠炎症,PTTG1 KO实验组小鼠的结肠黏膜上皮焦亡相关蛋白表达水平上调（P <0.05）。在HcoEpic中沉默PTTG1的表达,TNF-α刺激后,细胞的GSDMD蛋白表达水平上调（P <0...     

IL-21重组载体的构建及其诱导T细胞活化增殖的研究

目的构建小鼠IL-21基因真核重组表达载体,为IL-21基因治疗肿瘤奠定基础。方法分离BALB/c小鼠脾细胞,提取总RNA,经RT-PCR扩增IL-21基因片段,构建重组质粒pcDNA3.1(-)-IL-21,分组免疫小鼠,检测其脾细胞膜表面CD分子。结果重组质粒测序结果与GenBank中序列一致,大小为490 bp。核酸疫苗免疫组小鼠脾细胞表面CD分子表达量明显高于PBS和pcDNA3.1(-)空质粒组。结论成功的构建了重组质粒pcDNA3.1(-)-IL-21,该核酸疫苗免疫小鼠后,可有效地诱导其体内T细胞的活化增殖。     

系统性红斑狼疮患者血清白细胞介素2、6、10水平的变化及临床意义

系统性红斑狼疮(systemic lupus erythematosus,SLE)是以细胞免疫调节异常、多克隆B细胞活化为主要特征的自身免疫性疾病,为了解本病T细胞亚群的偏移情况,我们检测了SLE患者活动期血清白细胞介素2(IL-2)、IL-6和IL-10水平,现将结果报告如下。     

促丝裂原活化淋巴细胞诱导抗双链NDA抗体生成

抗双链DNA(ds-DNA)抗体是系统性红斑狼疮(SLE)的特征性和致病性抗体,但该自身抗体的诱生机理至今不明.我们用刀豆蛋白(Con A)和脂多糖(LPS)活化的小鼠淋巴细胞为自身抗原,分别免疫同系小鼠,均能诱导出IgG类抗ds-DNA抗体,并且小鼠肾脏有IgG类免疫复合物沉积形成.结果提示活化淋巴细胞的核内成分是诱生抗ds-DNA抗体的自身抗原.     

中期因子对实验性脑脊髓膜炎小鼠自身反应性Th17细胞活化的影响

目的观察中期因子对实验性脑脊髓膜炎小鼠自身反应性Th17细胞活化的影响。方法利用RT-PCR方法检测小胶质细胞系6-3细胞前炎症细胞因子IL-1β、IL-6和TNF-α及炎性介质i NOS m RNA表达,ELISA方法检测培养上清中上述细胞因子的表达水平,Greiss方法检测6-3细胞培养上清中NO水平;使用C57BL/6小鼠建立实验性脑脊髓膜炎小鼠动物模型,免疫磁珠纯化CD4+T细胞,与6-3细胞共培养,ELISA方法检测培养体系中培养上清IL-17的水平。结果与生理盐水对照相比,中期因子以剂量依赖方式促进6-3细胞表达IL-1β、IL-6和TNF-α及分泌NO的水平(P<0.05),中期因子适体抑制上述细胞因子及NO的表达(P<0.05)。体内实验结果表明中期因子适体抑制实验性脑脊髓膜炎小鼠MOG35-55特异性Th17细胞产生IL-17的水平(P<0.05)。结论中期因子通过活化小胶质细胞促进自身反应性Th17细胞活化。中期因子适体抑制小胶质细胞活化及实验性脑脊髓膜炎小鼠MOG35-55特异性Th17细胞活化。     

狼疮样肾炎小鼠脾细胞钙调磷酸酶活性检测及Th2细胞因子表达变化

目的:检测狼疮样肾炎小鼠脾细胞中钙调磷酸酶(CaN)活性,探讨CaN与肾炎小鼠脾细胞IL-6、IL-10表达的关系。方法:采用慢性移植物抗宿主小鼠(GVHD)模型,CaN活性检测采用发色底物法。结果:(1)模型组血清抗ds-DNA抗体BI值显著升高,与正常组比差异有显著性(P<0.05);FK506组BI值与正常组比较,差异无显著性。(2)狼疮样肾炎小鼠脾脏中CaN活性与血清中抗ds-DNA抗体呈正相关关系(r=0.925,P<0.01)。(3)脾细胞自身增殖反应性模型组明显高于正常组(P<0.05),FK506组与正常组比较,差异无显著性(P>0.05);FK506显著抑制刀豆蛋白(ConA)诱导的脾细胞增殖反应性,与模型组比较,差异有显著性(P<0.05)。(4)模型组脾细胞中CaN活性显著增强,与正常组比较差异有显著性(P<0.05);FK506组脾细胞CaN活性明显降低,与模型组比较差异有显著性(P<0.05)。(5)模型组小鼠脾细胞ConA诱导分泌Th2细胞因子IL-6、IL-10水平显著高于正常组及FK506组。结论:狼疮样小鼠脾细胞存在CaN过度活化,Th2细胞因子高表达与其CaN过度活化密切相...     

小鼠miR-144/451降低红细胞活性氧簇水平抑制AKT蛋白磷酸化的实验研究

目的探讨miR-144/451、活性氧簇(ROS)与AKT磷酸化的关系。方法流式细胞仪检测野生型小鼠(WT)与miR-144/451基因敲除(KO)小鼠的外周血红细胞中ROS水平。Western blot检测WT小鼠与miR-144/451 KO小鼠的骨髓有核红细胞、红系细胞系G1E细胞中磷酸化AKT(p-AKT)水平。结果与WT小鼠比较,miR-144/451 KO小鼠外周血红细胞中ROS水平显著增高(P 0. 05)。miR-144/451 KO小鼠骨髓有核红细胞中p-AKT的表达水平高于WT小鼠(P 0. 05)。ROS的升高是引起AKT磷酸化的主要原因,14-3-3ζ表达增加是引起AKT磷酸化的次要原因。结论 miR-144/451通过降低红细胞中ROS水平来抑制AKT蛋白磷酸化,从而使得红细胞免受氧化攻击。     

Cytokines and their receptors as biomarkers of systemic lupus erythematosus

Systemic lupus erythematosus is the most clinically diverse autoimmune disease. Owing to its heterogeneous presentation, clinical management of systemic lupus erythematosus remains as one of the greatest challenges. Therefore, there is a great need to assess disease activity accurately. Biomarkers can be objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention, and may also predict the risk of the disease, confirm diagnosis, monitor disease activity and provide prognostic information. Cytokines play an important and diverse role in the immune dysregulation in systemic lupus erythematosus. Measuring serum levels of soluble IL-2 receptor, IL-6, IL-10, soluble TNF receptor and IFN-alpha/IFN-induced genes may be promising biomarkers of disease activity in systemic lupus erythematosus.     

Comparison of different antinucleic acid antibody spectrotypes in spontaneous, induced, and murine lupus.

Sera from patients with systemic lupus erythematosus and from mice spontaneously developing lupus were subjected to isoelectric focusing by a microsucrose gradient method. The spectrotypes of human antibodies to native DNA, denatured DNA, and polyriboadenylic acid (poly A) were compared. Antibodies to native DNA and denatured DNA focused into two regions whose boundaries were pH 5.0-7.0 and pH 8.5-10.0. Antinative DNA antibodies were more homogeneous than antidenatured DNA antibodies. Anti-DNA antibodies in cryoglobulins showed more restriction than those present in serum. There was no relationship between spectrotype and pattern of disease expression. Murine antibodies to native DNA were more heterogeneous than human anti-DNA antibodies. The spectrotypes of antidenatured DNA antibodies from patients with systemic lupus erythematosus or drug induced lupus, or from an immunized rabbit, were similar. Likewise, antibodies to poly A were similar in both human and murine lupus. In contrast to anti-DNA, antibodies to poly A were restricted and focused only in the alkaline range (pH 9.5-10.0). The spectrotype of antipoly A antibodies induced by lipopolysaccharide were comparable but had an additional small band at pH 6.2. Our results suggest unique antibody spectrotypes with varying degrees of restriction for different nucleic acid antigens. Furthermore, spontaneous and induced autoantibodies have similar spectrotypes. Thus, the B cell clones producing antinucleic acid antibodies may be similar whether they are activated spontaneously, following immunization, or as a consequence of polyclonal stimulation.     

High interleukin-10 production in first-degree relatives of patients with generalized but not cutaneous lupus erythematosus.

BACKGROUND: Preclinical research suggests that interleukin-10 (IL-10) is associated with susceptibility to and severity of systemic lupus erythematosus. Chronic cutaneous lupus erythematosus is thought to be immunogenetically different from systemic lupus erythematosus. We hypothesized that high innate production of IL-10 underlies systemic but not chronic cutaneous lupus erythematosus. METHODS: IL-10 production was determined after endotoxin stimulation of whole-blood samples. In whole-blood samples, disease activity and medication influence the IL-10 production in patients. Therefore, healthy first-degree relatives of patients were evaluated. One hundred sixty-six first-degree relatives of patients with systemic lupus, 50 first-degree relatives of patients with chronic cutaneous lupus, and 133 control persons were studied. Innate IL-10 production of the patient was estimated as the mean IL-10 production of the unaffected relatives. Polymorphisms located -1082, -819, and -592 base pairs relative to the IL-10 gene were typed by allele-specific oligohybridization of polymerase chain reaction-amplified DNA fragments. RESULTS: IL-10 production was higher in the families of patients with systemic lupus than in the control families (1517 +/- 94 vs 1180 +/- 59 pg/mL; P = 0.003). IL-10 production in the families of patients with chronic cutaneous lupus was similar to that in control families (1216 +/- 82 vs 1180 +/- 59 pg/ml; P = 0.74). IL-10 production was also similar in families of patients with severe compared with nonsevere systemic lupus (P = 1.0). The frequency of -1082/-819/-592 haplotypes GCC, ACC, and ATA was similar among patients and compared with the control persons (P = 0.29). CONCLUSIONS: High innate IL-10 production underlies susceptibility for systemic lupus erythematosus but not the severity of the disease. It is not related to chronic cutaneous lupus erythematosus.     

Immunoassay with fluorescein-labelled antigens--device of a method and its clinical application.

A simple, sensitive, highly reproducible method for assay of antibodies using fluorescein-labelled antigens according to the method by Farr is described. We measured titers of anti-BSA antibodies and antigen binding capacity of sera in BSA immunized rabbits by fluorometric assay with fluorescein isothiocyanate (FITC) labelled BSA, and obtained highly reproducible and quantitative results. The resultd times more sensitive than the results from single radial immunodiffusion. FITC-labelled double or single stranded calf thymus DNA was incubated with sera of patients with systemic lupus erythematosus and New Zealand Black mice. Both anti-double and single stranded DNA antibodies were detected in sera of systemic lupus erythematosus, but only anti-single stranded DNA antibodies in sera of New Zealand Black mice.     

微小RNA调控干扰素-α在系统性红斑狼疮发病机制中的作用

系统性红斑狼疮是一种多系统受累的自身免疫病。干扰素-α作为系统性红斑狼疮免疫紊乱的关键因素, 对其作用机制及信号通路的研究可进一步揭示系统性红斑狼疮的发病机制, 并为该病的临床治疗提供新策略。近年研究发现, 微小RNA在系统性红斑狼疮发病机制中具有重要作用, 且微小RNA异常表达参与Ⅰ型干扰素通路的调节。本文综述了微小RNA对Ⅰ型干扰素通路的调节及其在系统性红斑狼疮发病机制中的作用, 对进一步认识系统性红斑狼疮的发病机制具有重要意义。     

系统性红斑狼疮小鼠骨髓间充质干细胞的成骨、成脂分化能力下降

背景：系统性红斑狼疮患者的骨髓间充质干细胞与正常人相比是否有不同,相关报道较少。 目的：对比系统性红斑狼疮模型小鼠与正常小鼠的骨髓间充质干细胞多向分化能力的差别。 方法：分离培养系统性红斑狼疮模型小鼠与C57BL小鼠的骨髓间充质干细胞（对照组）,分别进行成骨、成脂诱导分化,观察两种小鼠的骨髓间充质干细胞的分化能力。 结果与结论：C57BL小鼠的骨髓间充质干细胞经传代后,为长梭形,呈均匀分布生长;系统性红斑狼疮模型小鼠的骨髓间充质干细胞表现为生长缓慢,细胞相对C57BL小鼠要少一些。经过成骨诱导显示系统性红斑狼疮模型小鼠的钙结节和钙盐明显少于对照组,PCR检测成骨基因Runx2,碱性磷酸酶,骨钙素表达也明显下降。经过成脂诱导显示系统性红斑狼疮模型小鼠的脂滴明显少于对照组,PCR检测成脂基因PPARγ2,脂蛋白酯酶表达也明显下降。说明系统性红斑狼疮模型小鼠的骨髓间充质干细胞成骨与成脂能力都低于C57BL小鼠,系统性红斑狼疮模型小鼠的骨髓间充质干细胞的多向分化能力受损。     

Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity

Autoantibodies from many patients with systemic lupus erythematosus bind the Sm autoantigen B/B' polypeptide. The binding of serial serum specimens to the 233 overlapping octapeptides of Sm B/B' have shown that of the B/B'-derived octapeptides, PPPGMRPP and PPPGIRGP are early targets of the autoimmune response in some lupus patients. Rabbits immunized with PPPGMRPP and PPPGIRGP develop antibodies which not only bind these octapeptides, but also subsequently bind many other octapeptides of Sm B/B'. Eventually, the rabbits immunized with one octapeptide develop autoantibodies that bind other spliceosomal proteins including D, 70K, A, and C. Any mechanisms that operate to maintain tolerance or anergy for the spliceosome are thus overcome. Features considered typical of human systemic lupus erythematosus are also found in these peptide-immunized animals, such as antinuclear antibodies, anti-Sm precipitins, anti-double-stranded DNA, thrombocytopenia, seizures, and proteinuria. This disease model provides access to a mechanism for the development of humoral autoimmunity and may provide a basis to explain the immunopathogenesis of lupus in humans.     

Anti-inflammatory effect of MAPK phosphatase-1 local gene transfer in inflammatory bone loss

Alveolar bone loss associated with periodontal diseases is the result of osteoclastogenesis induced by bacterial pathogens. The mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a critical negative regulator of immune response as a key phosphatase capable of dephosphorylating activated MAPKs. In this study, rat macrophages transduced with recombinant adenovirus (Ad.)MKP-1 specifically dephosphorylated activated MAPKs induced by lipopolysaccharide (LPS) compared with control cells. Bone marrow macrophages from MKP-1 knockout (KO) mice exhibited higher interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and select chemokine compared with wild-type (WT) mice when stimulated by LPS. In addition, bone marrow cultures from MKP-1 KO mice exhibited significantly more osteoclastogenesis induced by LPS than when compared with WT mice. Importantly, MKP-1 gene transfer in bone marrow cells of MKP-1 KO mice significantly decreased IL-6, IL-10, TNF-α and chemokine levels, and formed fewer osteoclasts induced by LPS than compared with control group of cells. Furthermore, MKP-1 gene transfer in an experimental periodontal disease model attenuated bone resorption induced by LPS. Histological analysis confirmed that periodontal tissues transduced with Ad. MKP-1 exhibited less infiltrated inflammatory cells, less osteoclasts and less IL-6 than compared with rats of control groups. These studies indicate that MKP-1 is a key therapeutic target to control of inflammation-induced bone loss.     

Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. Association of high avidity antinative DNA antibody with glomerulonephritis.

Significant differences in both specificity and avidity of anti-DNA antibodies were observed in the sera of groups of patients with active systemic lupus erythematosus glomerulonephritis, active systemic lupus erythematosus without nephritis, and in IgG eluates obtained by DNAase digestion of isolated glomeruli from glomerulonephritic kidneys. With methylated albumin-kieselguhr fractionated 3H-HeLa DNA as a source of native or single-strand DNA antigen in a modified Farr assay, an increased level of antibody to native DNA was associated with active systemic lupus erythematosus, particularly active nephritis. The avidity of antinative DNA estimated from plots of the reciprocals of bound and free antigen according to the Sips distribution formula was significanly lower in active glomerulonephritis sera than in sera from patients with active systemic lupus erythematosus without nephritis. However, antinative DNA of uniformly high avidity was found in the glomerular eluates. Avidity of single-strand DNA antibodies did not differ in the various patient groups. The data stronly supprot a major role for high avidity antinative-DNA in DNA/antiDNA immune complex-induced glomerular injury in systemic lupus erythematosus.     

A novel isoform of the Ly108 gene ameliorates murine lupus

Studies of human systemic lupus erythematosus patients and of murine congenic mouse strains associate genes in a DNA segment on chromosome 1 with a genetic predisposition for this disease. The systematic analysis of lupus-prone congenic mouse strains suggests a role for two isoforms of the Ly108 receptor in the pathogenesis of the disease. In this study, we demonstrate that Ly108 is involved in the pathogenesis of lupus-related autoimmunity in mice. More importantly, we identified a third protein isoform, Ly108-H1, which is absent in two lupus-prone congenic animals. Introduction of an Ly108-H1-expressing transgene markedly diminishes T cell-dependent autoimmunity in congenic B6.Sle1b mice. Thus, an immune response-suppressing isoform of Ly108 can regulate the pathogenesis of lupus.