SLE模型新西兰小鼠高IgG血症易感基因的定位

目的：定位自发性ＳＬＥ模型００（ＮＺＢｘＮＺＷ）Ｆ１小鼠高ＩｇＧ血症的遗传易感基因。方法：建立（ＮＺＢｘＮＺＷ）Ｆ１ｘＮＺＷ回交小鼠模型,采用覆盖小鼠１９条染色体的多态性微卫星遗传标记及数量性状位点（ＱＴＬ）分析进行基因定位。结果：高ＩｇＧ血症易感基因与小鼠第１条染色体末端９２．３ｃＭ处微卫星遗传标记ＤＩＭｉｔ３６肯定连锁（Ｌｏｄｓ值〉３）,该位点附近９２．０ｃｍ处存在Ｆｅｇｒ２ｂ基因,且回交小鼠     

SLE模型小鼠高IgG血症易感基因-Fcgr2b基因突变及对其表达的影响

目的:测定系统性红斑狼疮(SLE)小鼠模型 (NZB×NZW)F1双亲NZB ,NZW小鼠Fcgr2b基因启动子区核酸序列及其表达的改变,明确Fcgr2b基因的突变性质、对该基因表达的影响及与高IgG血症的关系。方法:采用核酸序列分析测定NZB ,NZW小鼠Fcgr2b基因启动子区突变性质;RT PCR检测Fcgr2b基因表达的改变;ELISA法测定比较(NZB×NZW)F1,NZB和NZW小鼠及(NZB×NZW)F1×NZW回交小鼠Fcgr2b基因B/W型与W /W型组间血清总IgG水平。结果:NZB小鼠Fcgr2b基因启动子区与正常鼠BALB/c相比存在2个部位碱基缺失,分别为13bp及3bp。NZW小鼠除有3个碱基置换外,与BALB/c鼠该基因启动子区序列相同;NZB小鼠Fcgr2b基因mRNA的表达较正常鼠BALB/c降低;NZB小鼠血清总IgG水平明显高于NZW小鼠(P <0 0 5 ) ,回交小鼠Fcgr2b基因B/W型组血清总IgG水平明显高于W/W型组(P <0 0 0 0 1)。结论:NZB小鼠Fcgr2b基因启动子区存在碱基缺失,且该缺失突变可引起其表达的降低而导致高IgG血症     

CD4^＋CD45^＋T细胞对SLE病人B细胞产生自身抗体的调节作用

选择体外抗ＤＮＡ，抗ＣＤＬ抗体双阳性的ＳＬＥ病人Ｂ细胞做为靶细胞，分别与不同的ＳＬＥ病人或正常人ＣＤ４＾＋ＣＤ４５＾＋Ｔ细胞亚群共同培养，结果显示，ＳＬＥ病人ＣＤ４＾＋ＣＤ４５ＲＯ＾＋Ｔ细胞对自身抗体的产生有明显的辅助作用，而ＣＤ４＾＋ＣＤ４５ＲＡ＾＋Ｔ细胞的作用不明显。     

13种自身抗体检测对SLE的诊断价值及临床意义

目的探讨13种自身抗体对系统性红斑狼疮（SLE）的诊断价值和临床意义。方法抗核抗体（ANA）的检测采用间接免疫荧光法;抗ds-DNA抗体的检测采用快速金标渗滤法;11种可抽提核抗原（ENA）抗体的检测采用免疫印迹法。结果（1）223例SLE患者抗核抗体谱中,ANA、ds-DNA及抗ENA抗体SS-A/Ro60、SmD1、SS-A/Ro52和U1-RNP的阳性率较高,分别为95.06%、52.91%、50.22%、27.35%、24.66%和21.08%;Jo-1和P0抗ENA抗体的阳性率较低,分别为1.79%和0.90%;正常对照组仅ANA有5%的阳性率,其余抗体均为阴性。（2）抗体阳性的患者主要集中在10～50岁之间,10岁以下和50岁以上的患者比较少,平均年龄35岁。（3）SLE患者ANA-LIA的检测大多数患者的条带在4条以内,随着条带的增多,患者的数量减少。（4）抗ds-DNA抗体与抗SmD1抗体、抗核小体抗体的阳性率有显著差异（P〈0.01）,与抗SS-A/Ro60抗体的阳性率比较无显著差异（P〉0.05）。抗SmD1抗体与抗核小体、抗SS-A/Ro60抗体的阳性率有显著差异（分别为P〈0.05和P〈0.01）,与抗U1-RNP抗体的阳性率无显著差异（P〉0.05）。（5）104例SLE患者的血清免疫球蛋白和补体水平的异常,主要体现在IgG和C3异常。结论自身抗体的检测对SLE的诊断、治疗和疗效观察具有重要意义,多项自身抗体联合检测有利于提高SLE的免疫学诊断的阳性率。     

SLE病人外周血Bcl—2／JH基因重排检测的意义

应用聚合酶链反应（ＰＣＲ）方法检测３１例ＳＬＥ病人外周血单个核细胞（ＰＢＭＣ）Ｂｃｌ－２／ＪＨ基因重排现象和流式细胞仪间接双标记法分析其Ｔ（ＣＤ３）、Ｂ（ＣＤ１９）细胞Ｂｃｌ－２蛋白的表达。结果显示，ＳＬＥ病人Ｔ细胞Ｂｃｌ－２蛋白表达明显高于正常人（４２．９５％±２８．４７％对比９．９４％±４．９６％，Ｐ＝０．０００４），尤其以活动期ＳＬＥ病人为明显，而Ｂ细胞Ｂｃｌ－２蛋白表达与正常人之间并无统计学差异（７９．２１％±１０．６９％对比８１．９６％±６．９７％；Ｐ＝０．４６０２）。７例ＳＬＥ病人具有典型的Ｂｃｌ－２／ＪＨ基因重排（占２２．５８％），且均为ＳＬＥ活动期病人，其Ｔ细胞Ｂｃｌ－２蛋白表达明显高于无基因重排的ＳＬＥ病人，其Ｂ细胞Ｂｃｌ－２表达并无差异（Ｐ＞０．３９０５）。说明Ｂｃｌ－２／ＪＨ基因重排现象可见于ＳＬＥ，并与Ｔ细胞Ｂｃｌ－２蛋白高表达有关，表明细胞凋亡抑制基因Ｂｃｌ－２在ＳＬＥ发病机制中具有重要作用。     

小鼠IgG2b单克隆抗体Fab、Fc和Fab／c片段的制备和纯化（文摘）

本文报道了用小鼠IgG2b单克隆抗体制备和纯化Fab、Fc和Fab／c片段。由于IgG2b抗体具有对蛋白酶敏感的特性，用胃蛋白酶在酶与抗体之比为1：30，pH4．8的条件下消化抗体．可获得的Fab／c片段产量高达30％。消化液经DEAE—纤维素初步层析，可以含有Fc片段的混合液中分离出Fab片段。     

伴CD11b突变的SLE患者干扰素诱导基因的表达研究

为探讨SLE患者CD11b rs1143679突变与WT的临床特征及干扰素诱导基因(IFN-stimulated gene,ISG)的表达差异,收集1 193例SLE患者,进行CD11b rs1143679单核苷酸多态性检测,分析其临床表现;采用荧光定量PCR检测突变和部分WT患者IFIT1、IFIT4、ISG15在mRNA和蛋白质水平的表达,1 193例患者CD11b突变频率为1.84%(n=22)。结果显示,两组患者蛋白尿的发生差异具有统计学意义(P=0.032),其中突变患者24h尿蛋白值为(0.61±0.18)g;WT患者为(0.39±0.14)g(P=0.034);突变患者发生肾损伤的概率为68.2%,较WT患者显著增加(47.3%)(P=0.042);突变患者补体C3水平也较WT患者明显降低(P=0.021)。CD11b突变患者PBMC中IFIT1(P=0.025)、IFIT4(P=0.031)、ISG15(P=0.030)mRNA水平明显高于WT患者并与蛋白尿的发生呈正相关,其蛋白质相对表达量显著高于WT患者(P<0.05)。综上,CD11b突变的SLE患者肾损伤风险增加,ISG表达上调,可能在SLE发生发展中发挥重要作用。     

大肠杆菌DNA对BXSB小鼠的抗dsDNA抗体产生的影响

抗dsDNA抗体是系统性红斑狼疮(SLE)的血清学标志,其产生机制尚不清楚。CpG基序是细菌、病毒等微生物基因组内含有的以CG为核心的功能片段,体内外证实CpG基序具有强烈的免疫激活功能。本实验评价了CpG对BXSB狼疮小鼠产生抗dsDNA抗体的影响,旨在为SLE的治疗学和发病学提供新的线索。提取含有丰富CpG基序的大肠杆菌DH5α(由武汉大学生命科学院胡远扬教授惠赠)DNA ,经S1核酸酶纯化,纯化的dsDNA给予HpaⅡ内切酶消化,然后进行CpG序列分析。将纯化的大肠杆菌dsDNA (nECDNA5 0 μg)、小牛血清白蛋白(75 μg)和完全弗氏佐剂(CFA …     

GM-CSF对抗原化抗体基因免疫TH细胞应答的调节作用

目的研究GM-CSF(粒-单核巨噬细胞集落刺激因子)与抗原化抗体联合基因免疫时,GM-CSF对TH细胞应答的调节作用。方法在编码免疫球蛋白重链的质粒中分别克隆黏蛋白1(MUC1)中特异性PDTRP抗原表位和GM-CSF编码基因,构建含PDTRP和GM-CSF编码基因的抗原化抗体表达重组体。经脾免疫和肌肉加强的方式免疫BALB／c小鼠。采用ELISA方法检测小鼠血清中特异性抗体的水平及其亚类并动态观察;RT-PCR方法检测TH细胞分化中细胞因子和转录因子的表达水平。结果抗原化抗体基因免疫能诱导机体产生免疫应答。GM-CSF增强抗原化抗体基因免疫诱生的抗PDTRP特异性IgG抗体水平并且伴随IgG1／IgG2a显著性升高,同时可增强淋巴细胞TH2型细胞因子及转录因子(IL-4、GATA-3)mRNA的表达水平。结论GM-CSF在增强抗原化抗体基因免疫诱导的免疫应答的同时使得免疫应答向TH2方向偏移。     

IgG型抗双链DNA抗体的产生机制与致病性研究

IgG型抗双链DNA抗体是诊断系统性红斑狼疮（SLE）的重要自身抗体之一。该抗体的产生过程受紫外线、微生物感染等外源性诱导因素和自身基因易感性机制的调控,细菌DNA中高度保守的CpG序列在抗双链DNA抗体产生过程中起免疫佐剂的作用,且该过程需要Toll样受体9（TLR9）与IL-10等因素的参与。IgG型抗双链DNA抗体与狼疮肾炎关系非常密切,通过与肾脏固有细胞表面的某些靶抗原如α辅肌动蛋白等发生交叉结合可能是其致病机制之一。但抗双链DNA抗体阴性患者也可出现特异性肾脏损害,提示双链DNA可能并非激活抗体反应的唯一抗原。     

Cross-reactivity of IgG anti-DNA-secreting B cells in patients with systemic lupus erythematosus

This study is the first to analyze the cross-reactivity of in vivo activated B cells from patients with systemic lupus erythematosus. A chamber ELIspot assay was used to determine whether lymphocytes secreting antibodies that bound to DNA or 2,4,6-trinitrophenol (TNP)-keyhole limpet-hemocyanin (KLH) could simultaneously bind to the unrelated antigens actin or ovalbumin. IgM anti-DNA-, IgM anti-TNP-KLH- and IgG anti-TNP-KLH-secreting B cells from patients and controls showed similar levels of cross-reactivity (ranging from 6% to 23%, depending upon the antibody isotype and antigen pair examined). In general, IgG-producing cells were less cross-reactive than IgM producers from the same individual (on the average threefold, p < 0.001). In contrast, IgG anti-DNA-secreting B cells from lupus patients (i) showed no decrease in cross-reactivity when compared to IgM anti-DNA-secreting cells and (ii) were significantly more cross-reactive than control IgG anti-DNA-secreting cells and IgG anti-TNP-KLH secreting cells from patients (p < 0.001). The degree of IgG anti-DNA cross-reactivity correlated with disease activity (r = 0.52, p < 0.02). The implications of these findings with respect to repertoire expression and disease pathogenesis are discussed.     

系统性红斑狼疮患者血清泌乳素水平的检测及其临床意义

目的：探讨系统性红斑狼疮(SLE)患者血清泌乳素(PRL)水平检测的临床意义。方法：应用免疫放射量度分析法测定了75例SEE患者与25例健康人血清PRL水平。结果：SLE患者血清PRL水平明显高于正常对照组，且活动期升高更明显；高泌乳素血症(HPRL)发生率为40％，伴HPRL的患者肾损害发生率明显高于血清PRL正常者；血清PRL水平与SLEDM评分、抗ds-DNA抗体滴度倒数呈正相关，与C3呈负相关。结论：SLE患者血清PRL水平升高与病情活动相关，其检测可作为监测狼疮病情活动性的指标之一；血清PRL水平升高与肾脏损害相关，提示PRL可能在SLE肾损害中起作用。     

抗核小体抗体与儿童系统型红斑狼疮的相关性

目的探讨抗核小体抗体（AnuA）在诊断儿童系统型红斑狼疮（SLE）中的敏感性和特异性，了解AnuA与儿童SLE临床特征、疾病活动性的相关性。方法用Hep-2细胞提取核小体。用酶联免疫吸附方法（ELISA）测定血清中的AnuA，分析AnuA和临床表现的相关性。结果52例SLE中18例AnuA阳性。27例疾病对照组中1例AnuA阳性，30例正常对照组AnuA均为阴性。AnuA在儿童SLE中的敏感性为35％，特异性为96％。AnuA阳性组SLE患儿100％有中枢神经系统损害，AnuA阴性组为47％（x^2=14．57，P〈0．05）。AnuA阳性组SLE患儿100％有肾脏损害，AnuA阴性组为5l％（x^2=13．31，P〈0．05）。AnuA阳性和AnuA阴性的SLE患儿SLEDAI评分高于10分者分别为100％和71％（x^2=6．56，P〈0．05）。结论AnuA对儿童SLE的诊断具有较高的特异性，有助于抗dsDNA抗体、抗心磷脂抗体阴性的儿童SLE的诊断。     

Placental pathology in systemic lupus erythematosus with antiphospholipid antibodies

Systemic lupus erythematosus (SLE) is associated with a poor pregnancy outcome. Antiphospholipid antibodies (APL), which include lupus anticoagulant (LAC) and anticardiolipin antibodies (aCL), are frequently found in patients with SLE, and their presence has been associated with fetal loss. To examine placental pathologic features of SLE patients with APL, we performed a pathologic study on 47 placental tissue samples from 47 pregnant SLE patients with APL (15 patients; four LAC single-positive patients, seven aCL single-positive patients, four LAC and aCL double-positive patients) and without APL (32 LAC and aCL double-negative patients). The incidence of extensive infarction, decidual vasculopathy, decidual thrombosis and perivillous fibrinoid change, which have been thought to be characteristic lesions of APL placenta, was significantly higher in the LAC and aCL double-positive patients than in the patients without APL. Conversely, the above-mentioned lesions between the LAC or aCL single-positive patients and the APL negative patients did not differ significantly. Among the 15 patients with APL, two of the three patients with both decidual vasculopathy and thrombosis had extensive infarction associated with fetal death. Moreover, the patients having fetal death showed LAC and aCL double-positivity. In conclusion, this study indicated that the LAC and aCL double-positivity is an important factor for extensive infarction resulting from decidual vasculopathy and decidual thrombosis in the SLE placenta. Moreover, it was indicated that LAC and aCL double-positivity is an important risk factor for fetal death in the SLE patient.     

Antiribonuclease H2 antibodies are an immune biomarker for systemic lupus erythematosus

We previously reported that autoantibodies against the proliferating cell nuclear antigen protein (PCNA)-binding protein chromatin assembly factor-1 (CAF-1) are specifically found in patients with systemic lupus erythematosus (SLE). PCNA and its complex constituents elicit autoimmune responses in patients with SLE, suggesting that autoantibody diversification likely occurs owing to epitope spreading. Therefore, we sought to clarify whether patients with SLE exhibit an autoimmune response to Ribonuclease H2 (RNase H2), another PCNA-binding protein that regulates cell division. As results, RNase H2 autoantibodies were detected in the sera of 33.9% (19/56) of SLE patients, which was significantly higher than that observed in sera from other patients with systemic autoimmune diseases (polymyositis/dermatomyositis, systemic sclerosis, Sjogren’s syndrome, mixed connective tissue disease and rheumatoid arthritis) and healthy controls. Regression analysis also showed that serum anti-RNase H2 levels were strongly correlated to that of CAF-1 in SLE patients. Our data support the use of RNase H2 autoantibodies as a serum biomarker for SLE diagnosis. Moreover, the strong correlation observed between RNase H2 and CAF-1 suggests that intermolecular epitope spreading may play a critical role in autoantibody production and diversification in SLE.     

系统性红斑狼疮的分子遗传学研究

系统性红斑狼疮（systemiclupuserythematosus,SLE）是一种复合性多基因的自身免疫性疾病。遗传因素在SLE易感性方面起重要的作用。该文介绍目前已知几个与SLE的易感性有关的遗传基因。通过对这些基因研究,有利于阐明SLE的发病机制,进一步为SLE的预防、诊断和治疗提供重要的依据。     

IgG2 subclass restriction of anti-β2 glycoprotein 1 antibodies in autoimmune patients

The IgG subclass and light chain distribution of antiphospholipid antibodies (aPL) occurring in autoimmune patients were determined by means of two radioimmunoassays using either cardiolipinor β₂ glycoprotein 1 (β₂GP1)-coated microtitre plates and mouse MoAbs. Of 50 sera selected for positivity of anticardiolipin antibodies (ACA) of the IgG isotype, 32 (64%) possessed anti-β₂GPl antibodies and their presence was closely associated with clinical features of the antiphospholipid syndrome. Good correlations were found between ACA and anti-β₂GP1 antibodies when considering antibody level and patterns of light chain and IgG subclass, suggesting that, overall, the same antibodies were being measured. Light chain analysis showed the polyclonal origin of these antibodies and, in most sera, a trend towards use of λ chain. Among sera positive for anti-β₂GP1 antibodies, IgG2 was the major subclass reactive with β₂GP1 and cardiolipin (87% and 74β₂ of the IgG antibody activity, respectively). In contrast, in the group of 18 sera lacking anti-β₂GP1 antibodies, ACA were largely restricted to lgG3, with a lesser contribution by IgGl. A few selected sera from the anti-β₂GP1-positive group were shown to contain mixtures of antibodies that required β₂GP1 (restricted to IgG2 present in large amounts) and did not require this cofactor (restricted to IgG3 and/or IgG1 present in low amounts) for their reactivity with cardiolipin. There was no contribution of glycosylation to the epitopes recognized by anti-β₂GP1 antibodies, even though human anti-carbohydrate antibodies are restricted to the IgG2 subclass. These findings further emphasize the intra- and interindividual heterogeneity of aPL, and should help to discriminate clinically relevant specificies.     

Characterization of anti-endothelial cell antibodies in systemic lupus erythematosus (SLE).

IgG anti-endothelial antibodies (AEA), as measured by ELISA or immunoblotting technique could be detected in serum samples of 56 out of 64 patients with SLE (88%) and mainly occurred in monomeric form. AEA were not cell specific, because the binding reactivity was absorbed partially by both fibroblasts and peripheral blood mononuclear cells. No correlation was found between the presence of AEA and anti-nuclear antibodies. Immunoblotting revealed reactivity of AEA against endothelial antigens ranging in size from 15 to 200 kD. AEA titres were significantly higher in patients with joint or skin abnormalities, compared with patients without these abnormalities. A significant correlation was found between nephritis in SLE and the presence of AEA reactivity against endothelial membrane antigens of 38, 41 and 150 kD. These data show that the pattern of AEA reactivity in serum of SLE patients is heterogeneous, and suggest that AEA against a limited number of antigens may be involved in the pathogenesis of nephritis in SLE.