急性脑血管病患者协同刺激分子表达的研究

目的：探讨急性脑血管病患者血液中协同刺激分子白细胞分化抗原簇80（chuster of differention80,CED80,B7-1)、白细胞分化抗原簇86（cluster of differention86,CD86,B7-2)、白细胞分化抗原簇28（cluster of differention28,CD28）,细胞毒性T淋巴细胞抗原－4（cytotoxic T lymphocyte antigen-4,CTLA-4)蛋白的表达和B7－1、B7－2信使核糖核酸（messenger ribonucleic acid,mRNA)的表达其与急性脑血管病病损的关系。方法：应用分子生物学技术,检测急性期脑梗死（26例）,脑出血患者（26例）和年龄匹配的26名健康对照者外周血淋巴细胞B7－1,B7－2,CD28和CTLA－4蛋白的表达和外周血淋巴细胞B7－1、B7－2mRAN的表达,各病列均经头部CT或MRI并计算病灶体积,采用改良爱丁堡＋斯堪的那维亚评分标准（SSS）对临床神经功能 缺损程度进行评分,分析其相互关系。结果：外周血淋巴细胞B7－1、CD28和CTLA－4蛋白的表达,脑梗死组高于脑出血组,两组均高于健康对照组;外周血淋巴细胞B7－2蛋白的表达,三组间无显著性差异,健康对照组外周血淋巴细胞未检测到CTLA－4蛋白的表达;外周血淋巴细胞B7－1mRNA的表达,脑梗死组高于脑出血组,两组均高于健康对照,外周血淋巴细胞B7－2mRNA的表达,三组间无显著性差异,脑梗死组B7－1及CD28与SSS呈正相关,脑出血组协同刺激分子的表达以及病灶的大小与SSS无相关性,脑梗死组病灶的大小与SSS呈正相关,结论：协同刺激分子B7－1、CD28和CTLA－4在急性脑血管病的病理机制中起着重要作用。     

共刺激分子CD28：CTLA4/B7在实验性自身免疫性重症肌无力中的作用

目的 研究共刺激分子CD28:CTLA4/B7在实验性自身免疫性重症肌无力(EAMG)发病中的作用.方法 将雌性Lewis鼠随机分为EAMC组和对照组;EAMG组采用人工合成的Ra97-116肽段3次法免疫Lewis鼠,对照组同期注入等量的PBS;3次免疫接种后采用流式细胞术检测CD28、B7-2、B7-1、CTLA4在外周血、淋巴细胞、单核细胞中的表达.结果 EAMG组大鼠成模率75%;与对照组比较,外周血CD28、B7-2B7-1、CTLA4的表达明显增加(P<0.05～0.01);EAMG组大鼠外周血CD28、CTLA4主要在淋巴细胞表达及B7-1、B7-2在淋巴细胞、单核细胞表达显著增加(P<0.05～0.01).结论 EAMG大鼠存在共刺激分子CD28:CTLA4/B7表达异常,共刺激分子CD28:CTLA4/B7可能参与了EAMG的发生、发展.     

协同刺激分子在格林-巴利综合征患者 脑脊液、血及周围神经中的表达

目的　探讨协同刺激分子在格林 巴利综合征 (GBS)患者脑脊液、血和周围神经组织中的表达及其作用。方法　用RT PCR法及原位杂交法观察GBS患者外周血、脑脊液和周围神经组织中协同刺激分子B7 1、B7 2、CD2 8、CTLA 4的表达。结果　GBS患者外周血、CSF和周围神经组织中协同刺激分子CD2 8、B7 1、B7 2在炎性细胞上的表达明显高于对照组 ;GBS患者CSF中CD2 8、B7 1、B7 2mRNA的表达明显高于外周血。结论　在GBS发病过程中协同刺激分子的表达对T细胞的活化 ,进而导致体液免疫和细胞免疫反应起重要作用。     

协同刺激分子在实验性变态反应性神经炎发病中的作用及雷公藤多甙的影响

目的：探讨协同刺激分子在实验性变态反应性神经炎（EAN）发病中的作用及雷公藤多甙的影响。方法：用兔坐骨神经匀浆免疫小鼠建立EAN模型,雷公藤多甙（TWP）灌胃治疗,观察小鼠发病情况和病理改变;通过流式细胞计检测小鼠外周血淋巴细胞CD28,CTLA－4,B7－1,B7－2蛋白的表达,用RT－PCR检测外周血淋巴细胞B7－1和B7－2mRNA和表达。结果：EAN小鼠外周血淋巴细胞上协同刺激分子CD28,CTLA－4,B7－1,B7－2蛋白的表达水平明显增高,B7－1和B7－2 mRNA表达与蛋白的增加相平行,雷公藤多甙治疗组发病率及病变程度均明显降低。同时伴随CD28,B7－1,B7－2蛋白的表达及B7－1,B7－2,mRNA表达的水平降低。结论：协同刺激分子的表达对T细胞活化起重要作用;雷公藤多甙能减轻ENA的病变程度。可能与抑制了B7－1及B7－2的基因转录或转录以上环节和抑制了CD28翻译或翻译上环节有关。     

实验性自身免疫性重症肌无力大鼠CD28/CTLA-4:B7表达水平的研究

目的探讨实验性自身免疫性重症肌无力(EAMG)大鼠外周血单个核细胞(PBMC)CD28/CTLA4B7的表达水平。方法健康、雌性Lewis大鼠24只,随机分为正常组、EAMG组、完全福(氏)佐剂(CFA)对照组。EAMG组大鼠分别于足垫、腹部及背部皮下多点注射丁(氏)双鳍电鳐电器官乙酰胆碱受体蛋白乳剂1mL,第4周再次注射上述乳剂免疫大鼠。CFA对照组只接受等量的CFA皮下注射。初次免疫后7周分离PBMC,应用RTPCR和流式细胞术分析方法,分别进行CD28、CTLA4mRNA及B71、B72蛋白表达水平检测。结果(1)正常组大鼠PBMCCD28、CTLA4mRNA表达水平较低,尤其CTLA4mRNA仅有极少量表达;前二者在EAMG组大鼠表达水平均明显增加(P<0001),而正常组和CFA对照组之间表达水平差异无显著性(P>005)。(2)正常大鼠B71、B72在PBMC上仅少量表达而EAMG组表达明显增加(P<0001),正常组与CFA对照组比较差异均无显著性(P>005)。结论EAMG大鼠存在PBMCCD28/CTLA4B7协同刺激分子的表达异常,CD28/CTLA4B7共刺激通路可能参与了机体异常免疫反应的诱导与维持,在重症肌无力(MG)的发生过程中发挥重要作用。     

重症肌无力患者外周血T、B淋巴细胞Bcl-2的表达

目的　探讨重症肌无力 (MG)患者外周血单个核细胞Bcl 2蛋白表达及其临床意义。方法　以流式细胞仪双标记免疫荧光方法测定 4 7例临床确诊的MG患者外周血T、B淋巴细胞Bcl 2蛋白表达和CD3 T细胞Bcl 2蛋白表达的平均荧光强度 (MFI)。结果　 ( 1)MG组外周血CD3 、CD4 、CD8 T淋巴细胞和CD19 细胞Bcl 2蛋白表达明显高于对照组 (P <0 .0 1) ,CD3 T细胞蛋白表达Bcl 2的MFI( 0 .572± 0 .177)亦明显高于对照组 ( 0 .170± 0 .147) (P <0 .0 1)。 ( 2 )MG组外周血CD3 、CD4 、CD8 T淋巴细胞及CD19 细胞的Bcl 2蛋白表达与年龄无明显相关 ,而与临床严重程度绝对评分密切相关 (r=0 .63、0 .65、0 .61、0 .78,P <0 .0 5)。CD3 T细胞蛋白表达的MFI与MG患者病程相关密切 (r=0 .62 ,P <0 .0 1)。 ( 3)免疫抑制治疗后MG组临床严重程度绝对评分与淋巴细胞亚群Bcl 2蛋白表达、CD3 T细胞蛋白表达的MFI同步地较治疗前有明显下降 (P <0 .0 1)。结论　外周血淋巴细胞Bcl 2蛋白异常表达对MG发病及临床症状有重要作用。     

B7-H1分子免疫病理在多发性肌炎中的临床研究

目的 通过免疫组化染色了解协同刺激分子B7-H1蛋白在多发性肌炎(PM)和肢带型肌营养不良2B型(LGMD 28)患者肌组织中的表达情况,探讨其在PM诊断和鉴别诊断中的意义.方法 选择苏州大学附属第一医院神经内科自2006年1月至2009年12月收治的43例PM患者(PM组),26例LGMD 2B型患者(LGMD 2B组)及21例肌活检正常者(对照组).对所有成员行肌肉活检,冰冻切片后进行常规HE染色、免疫组织化学染色,检测肌组织中B7-H1蛋白的表达.结果 (1)PM组与LGMD 2B型组肌肉活检普通病理染色结果相似,表现为不同程度的坏死、吞噬、再生现象,伴有不同程度的炎细胞浸润.(2)PM组B7-H1蛋白阳性表达主要定位于细胞膜,呈棕黄色至棕褐色,主要集中在有炎细胞浸润的变性、坏死肌纤维上;其肌组织中B7-H1蛋白表达水平比较LGMD2B型组和对照组成员肌组织中水平明显增高(分别为69.77%、26.92%、4.76%),差异有统计学意义(P<0.05).结论 协同刺激分子B7-H1在PM患者肌组织中高表达,参与了PM的免疫学发病机制,可成为PM与继发性炎细胞浸润性肌病相鉴别的免疫病理标志.     

重症肌无力胸腺淋巴细胞亚群表型的变化

目的　研究重症肌无力 (myastheniagravis ,MG)患者胸腺的T、B淋巴细胞亚群表型 ,并分析与其外周血的相关性。方法　应用免疫荧光标记技术 ,经流式细胞仪分析 ,检测了 59例MG患者 ,包括 30例伴胸腺病变患者外周血的淋巴细胞亚群表型。另外 ,对 8例MG患者的胸腺和外周血经体外培养后进行检测。结果　 ( 1)MG患者外周血辅助性T细胞 (Th ,CD4 )异常增高 ,病程 6个月以内、伴胸腺增生者Th细胞均明显增多 ;( 2 )MG胸腺和外周血中经乙酰胆碱受体 (AChR)刺激后活化的Th细胞 (CD4 CD2 5 )、CD5-B细胞 (CD5- CD19 )明显增多 ,且外周血CD5-B细胞与自身血清乙酰胆碱受体抗体 (AChRAb)滴度显著相关 (P <0 .0 1) ,胸腺摘除术 (Tx)后 ,MG外周血CD4 CD2 5 、CD5- CD19 细胞均有所减少。结论　MG患者胸腺存在着异常的AChR特异应答性T、B淋巴细胞亚群表型 ,尤其以活化的Th细胞为著 ;CD5-B细胞的产生可能与MG密切相关。     

B7-H1分子免疫病理在多发性肌炎中的临床研究

目的 通过免疫组化染色了解协同刺激分子B7-H1蛋白在多发性肌炎(PM)和肢带型肌营养不良2B型(LGMD 28)患者肌组织中的表达情况,探讨其在PM诊断和鉴别诊断中的意义.方法 选择苏州大学附属第一医院神经内科自2006年1月至2009年12月收治的43例PM患者(PM组),26例LGMD 2B型患者(LGMD 2B组)及21例肌活检正常者(对照组).对所有成员行肌肉活检,冰冻切片后进行常规HE染色、免疫组织化学染色,检测肌组织中B7-H1蛋白的表达.结果 (1)PM组与LGMD 2B型组肌肉活检普通病理染色结果相似,表现为不同程度的坏死、吞噬、再生现象,伴有不同程度的炎细胞浸润.(2)PM组B7-H1蛋白阳性表达主要定位于细胞膜,呈棕黄色至棕褐色,主要集中在有炎细胞浸润的变性、坏死肌纤维上;其肌组织中B7-H1蛋白表达水平比较LGMD2B型组和对照组成员肌组织中水平明显增高(分别为69.77%、26.92%、4.76%),差异有统计学意义(P<0.05).结论 协同刺激分子B7-H1在PM患者肌组织中高表达,参与了PM的免疫学发病机制,可成为PM与继发性炎细胞浸润性肌病相鉴别的免疫病理标志.     

急性缺血性脑卒中患者外周血白细胞CD11b、CD18表达变化研究

目的　探讨急性脑卒中患者外周血白细胞黏附分子 CD1 1 b、CD1 8的表达 ,以及其在急性缺血性脑损伤发生、发展中的作用。方法　采用流式细胞术 ,用单克隆抗体标记定量测定 3 2例急性脑卒中患者发作 2 4h内外周血白细胞 CD1 1 b、CD1 8的表达量 ,以平均荧光强度 (MFI)的大小来表示 CD1 1 b、CD1 8的相对含量 ,在发病72 h、7d后再分别测定 1次。以 40例健康者作对照。结果　发病 2 4h内患者组白细胞表面黏附分子 CD1 1 b、CD1 8的表达显著高于对照组 (P <0 .0 1 ) ;在发病 72 h内患者组 CD1 1 b、CD1 8的表达逐渐降低 ,但仍高于对照组 (P<0 .0 5 ) ;发病 7d后 ,其结果与对照组相比差异无显著性 (P >0 .0 5 )。结论　急性脑卒中发病时 ,白细胞被激活 ,黏附分子 CD1 1 b、CD1 8表达上调 ,介导白细胞与内皮细胞黏附增强 ,可能会加重缺血后迟发性神经元死亡的病理生理过程。     

Expression of the costimulatory molecule BB-1 and its receptors in patients with scleroderma-polymyositis overlap syndrome

We investigated expression of costimulatory molecules BB-1, B7-1 (CD80), B7-2 (CD86), and their counter-receptors CD28 and CTLA-4 (CD152) in muscle biopsy specimens of patients with scleroderma-polymyositis overlap syndrome (SSc-PM), primary polymyositis (PM), and other related diseases to examine whether the muscle fibers in patients with SSc-PM behave as antigen-presenting cells (APCs). The major histocompatibility (MHC) class II-positive muscle fibers of SSc-PM patients reacted with monoclonal antibodies (mAb) against BB-1 but not against B7-1 or B7-2. The CD4+ T cells expressed the counter-receptors CD28 and CTLA-4, and bound with the BB-1-positive muscle fibers in cell-to-cell contact. Our findings show that muscle fibers in patients with SSc-PM function as "professional" APCs in a way distinct from muscle fibers in patients with primary PM.     

TGF-β1基因修饰的树突状细胞对实验性自身免疫性重症肌无力大鼠外周血单个核细胞CD28/CTLA-4:B7表达的影响

目的 探讨TGF-β1基因修饰的树突状细胞(DC)对实验性自身免疫性重症肌无力(EAMG)大鼠外周血单个核细胞(PBMC)CD28/CTLA-4:B7表达的影响.方法 近交系8～10周龄健康雌性Lewis大鼠30只,分为正常组、EAMG组、DC对照组、pcDNA3-TGF-β1-DC组、pcDNA3-DC对照组、生理盐水对照组.除正常组外,其余各组均采用丁氏双鳍电鳐电器官乙酰胆碱受体(AChR)蛋白二次免疫的方法复制EAMG大鼠模型.初次免疫后第5天分别皮下注射2×106的DC、pcDNA3-TGF-β1-DC、pcDNA3-DC及等体积的生理盐水,正常组和EAMG组不接受任何治疗.初次免疫后7周分离各组PBMC,应用RT-PCR和流式细胞术方法分别进行CD28、CTLA-4 mRNA及B7-1、B7-2蛋白表达水平的检测.结果 (1)正常组大鼠PBMC CD28、CTLA-4 mRNA的表达水平较低,尤其CTLA-4 mRNA极少量表达;EAMG组大鼠CD28、CTLA-4 mRNA的表达水平均明显增加;pcDNA3-TGF-β1-DC组CD28 mRNA的表达较EAMG组明显降低(P<0.01),而CTLA-4 mRNA的表达水平较EAMG组明显增加(P<0.05);EAMG组、DC治疗组、pcDNA3-DC对照组和生理盐水对照组之间CD28、CTLA-4 mRNA的表达水平无显著性差异(P＞0.05).(2)正常大鼠B7-1、B7-2在PBMC上少量表达;EAMG组B7-1、B7-2两者表达明显增加(P<0.001);pcDNA3-TGF-β1-DC治疗组B7-1、B7-2的表达较EAMG组均明显降低(P<0.01);DC对照组、pcDNA3-DC对照组、生理盐水对照组与EAMG组比较均无明显差异(P＞0.05).结论 EAMG大鼠存在PBMC CD28/CTLA-4:B7协同刺激分子的表达异常,主动调节机体的共刺激通路可能是TGF-β1基因修饰的DC治疗EAMG的机制之一.     

Expression of costimulatory molecules on peripheral blood mononuclear cells in multiple sclerosis

OBJECTIVE: To determine whether there was aberrant expression of costimulatory molecules and their receptors on peripheral blood mononuclear cells of MS patients and healthy individuals and whether expression correlated with disease status. METHODS: Forty-six patients with MS and 29 healthy individuals were analyzed by direct 2-color immunofluorescence flow cytometry for expression of CD80, CD86, CD28 and CTLA-4 on T and B cells and monocytes. RESULTS: Expression of CD80 on CD4+ T cells was upregulated in progressing MS patients compared to stable MS patients and controls. Marked increase in the expression of CD80 and CD86 was seen on both CD4+ and CD8+ T cells from an MS patient with rapidly progressing disease. No difference in the expression of the costimulatory molecules or their ligands was seen between IFN-beta treated and non-treated MS patients. CONCLUSIONS: Aberrant expression of costimulatory molecules occurs on T lymphocytes in MS patients with progressing disease.     

特发性多发性肌炎外周血淋巴细胞共刺激分子表达的研究

目的观察特发性多发性肌炎患者外周血淋巴细胞共刺激分子CD28、CD152、CD80、CD86表达,进一步分析共刺激分子与疾病的关系。方法研究分三组,即健康对照者（HC,n=5）、特发性多发性肌炎组（IPM,n=8）和肌营养不良组（MD,n=8）,后两组患者均经肌肉活检酶组织化学确诊。三组患者均通过流式细胞术检测外周血淋巴细胞CD4、CD8、CD4＋CD28＋、CD8＋CD28＋、CD152、CD80、CD86表达,并计算出CD4＋CD28-、CD8＋CD28-表达。结果与HC组比较,IPM组CD4＋CD28＋表达降低（31.9±6.6 vs 39.5±6.9,P〈0.05）,其所占CD4＋T细胞的百分率亦明显下降（92.2%±6.2% vs 98.1%±0.7%,P〈0.05）,与之相反的是CD4＋CD28-表达明显升高（2.0±1.8 vs 0.6±0.2,P〈0.05）。IPM组CD4、CD8表达及CD8＋CD28＋/CD8百分比与HC组无明显差异,CD8＋CD28-表达则呈上升趋势。IPM组CD152较HC组明显升高（18.5±13.8 vs 7.1±1.2,P〈0.05）,各组间CD80表达无明显差异,CD86以IPM组最高（6.8±1.9 vs 4.6±1.7,P〈0.05）。结论IPM患者外周血CD4阳性的T细胞表面的CD28表达下降,但CD152和CD86表达升高。     

The role of costimulation in autoimmune demyelination

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated, autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed costimulation, is required for complete T cell activation. The B7 family of cell surface molecules expressed on antigen presenting cells (APC) is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. Our studies have shown that costimulation provided by B7 molecules to its ligand CD28 is important in the initiation of the autoimmune response in EAE. Further, it appears the costimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the immune response. Previous work has shown that activated T cells and T cells of a memory phenotype are less dependent on costimulation than naive T cells. T cells reactive with myelin components that are involved in the pathogenesis of EAE and possibly MS would be expected to have been activated as part of the disease process. Building upon our prior work in the EAE model, we have tested the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished from naive myelin-reactive T cells by a lack of dependence upon costimulation for activation and that the costimulatory requirements of these myelin-reactive T cells change during the course of disease. Our studies in the EAE model have also addressed the mechanisms of extrathymic (peripheral) T cell tolerance following intravenous (i.v. ) administration of high dose antigen. It is believed that TCR signaling in the absence of costimulation is a vital component of peripheral tolerance mechanisms. However, recent evidence suggests that peripheral tolerance of antigen-specific T cells induced in vivo may require CTLA-4 engagement of the tolerized T cells. We have begun to examine the molecular mechanisms of tolerance induction following intravenous and intraperitoneal administration of myelin antigens in the EAE model and test the hypothesis that tolerance induction is dependent on the B7:CD28/CTLA-4 pathway. The results from our studies will enhance our understanding of the role that myelin-reactive T cells may play in the pathogenesis of MS. We have determined that MBP-reactive T cells in MS patients are less dependent upon CD28 costimulation than in normal controls, suggesting that these T cells were previously primed in vivo. Characterization of these CD28-independent myelin-specific T cells will have broad implications for a variety of immunologically based therapies in diseases such as MS.     

Differential expression of costimulatory molecules B7-1 and B7-2 on microglial cells induced by Th1 and Th2 cells in organotypic brain tissue.

Autoreactive T-cells are involved in demyelination, neurodegeneration, and the recruitment of peripheral macrophages and nonspecific activated T-cells in autoimmune diseases such as multiple sclerosis (MS). The ligation of costimulatory B7 molecules on microglia with CD28/CTLA-4 on T-cells is thought to be crucial to the onset and course of MS and its rodent model experimental autoimmune encephalomyelitis (EAE). It is currently unclear as to how far the nature of infiltrating T-cells has an impact on the expression of the B7 molecules on microglia, the resident antigen-presenting cells (APCs) of the brain. We studied the expression of B7-1 and B7-2 on microglia after encounter with preactivated Th1 and Th2 cells from transgenic mice whose T-cells express a receptor (TCR) either specific to myelin basic protein (MBP) or ovalbumin (OVA) using murine organotypic entorhinal-hippocampal slice cultures (OEHSC). Our main finding was that Th1 cells downregulate the constitutive expression of B7-2 and induce B7-1 expression while Th2 cells do not induce this B7-1 upregulation. The main difference between MBP- and OVA-specific cells was seen in experiments were Th1 cells had direct contact to APCs but not to brain tissue. In contrast to MBP-specific Th1 cells, OVA-specific Th1 cells required the addition of antigen to upregulate B7-1 and downregulate B7-2. When the cells were allowed to have contact to brain tissue, no difference was seen in the pattern of B7 regulation between OVA- and MBP-specific T-cells. Our data suggest that T-cells are able to modulate B7 expression on microglial cells in the brain independent of antigen presentation through TCR/MHC-II ligation but presumably by soluble mediators.     

T-cell costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are expressed in human microglia but not in astrocytes in culture

The B7-1 and B7-2 expressed on the 'professional' antigen-presenting cells (APC) of the lymphoid system are counterreceptors for the T cell antigens CD28/CTLA-4. The B7/CD28 interaction provides a critical costimulatory signal in the decision between functional activation or clonal anergy of T cells. To investigate the biological role of B7 in the central nervous system, constitutive and cytokine-induced expression of B7 was investigated in fetal human astrocytes and microglia in culture. B7-1 expression was minimally detectable in unstimulated microglia but was increased markedly following exposure to IFN-γ or GM-CSF. B7-2 was expressed at a high level in untreated microglia and upregulated to a small degree by exposure to IFN-γ or GM-CSF. In contrast, B7-1 and B7-2 were undetectable in astrocytes under unstimulated or IFN-γ/GM-CSF-treated conditions. These results indicate that both B7-1 and B7-2 are expressed in cultured human microglia but not in astrocytes.