AITD甲状腺细胞Fas、FasL的单抗免疫组化测定

测定凋亡相关蛋白Fas、FasL在自身免疫性甲状腺疾病(AITD)病人甲状腺细胞中的表达,以研究细胞凋亡在AITD发病中的作用.本研究采用甲状腺粗针穿刺取甲状腺组织,然后用单克隆抗体免疫组织化学法和图像分析检测甲状腺细胞中Fas、FasL的表达,于电镜下观察结果.Graves病(GD)组Fas、FasL染色强度显著低于对照组(P＜0.01).桥本氏甲状腺炎(HT)组两者染色强度显著高于对照组(P＜0.01).这说明Fas、FasL这两种凋亡相关蛋白在HT病人甲状腺细胞中表达显著增强,而在GD病人中表达较少,进一步证实了细胞凋亡在AITD发病机制中具有重要作用.     

自身免疫性甲状腺疾病胰岛素样生长因子及其结合蛋白的改变

为探讨胰岛素样生长因子 (IGF )、胰岛素样生长因子结合蛋白 (IGFBP )在自身免疫性甲状腺疾病 (AITD )中的变化及其影响 ,本研究检测了 5 6例AITD患者与 2 4例正常对照血清IGF 1、IGFBP 1～ 3及甲状腺功能 ,发现IGF 1在GD、HT与GD控制组明显低于正常对照 (P <0 0 1) ,IGFBP 1、IGFBP 2在GD组明显高于正常对照 (P <0 0 1,0 0 5 ) ,IGFBP 3在HT组明显低于正常对照 (P <0 0 5 ) ;IGF 1与甲状腺激素间无相关 ,IGFBP 1～ 3均与TT4相关 (r =0 34、 0 38、 0 31;P <0 0 5 )。提示机体甲状腺激素、免疫状态均可能影响IGF、IGFBP水平 ,而后者有可能参与调节AITD的进程。     

桥本甲状腺炎甲状腺组织细胞凋亡与凋亡基因Bcl—2、Bax表达

为了探讨桥本甲状腺炎 (HT)甲状腺组织细胞凋亡的变化、凋亡基因Bc1- 2、Bax的表达及其与HT发病机制的关系 ,采用TUNEL法检测 4 1例HT、10例正常甲状腺组织细胞凋亡状况 ,同时采用免疫组化S -P法检测Bc1- 2、Bax表达及分布 ,应用Mias99图像分析系统对免疫组化结果进行定量分析。结果表明 :HT组甲状腺细胞凋亡率及甲状腺组织Bc1- 2、Bax阳性颗粒面积、平均光密度和积分光密度均显著高于正常组 (P <0 .0 5 - 0 .0 0 1) ;HT甲状腺滤泡区Bc1- 2阳性颗粒面积和积分光密度与Bax的比值明显低于正常甲状腺的比值 ,其组织结构破坏较重区域中小滤泡及失去正常滤泡结构的腺上皮Bax表达高于其它腺上皮 ,滤泡结构较完整的区域Bc1- 2表达较高 ,提示甲状腺细胞凋亡增高是HT甲状腺滤泡结构破坏和功能异常的重要机制之一 ;凋亡基因Bc1- 2、Bax表达的失衡与HT时甲状腺细胞凋亡增高密切相关     

干扰素-γ对Graves病甲状腺细胞凋亡的影响

目的 探讨干扰素一γ(interferon-γ,IFN-γ)对Graves病(GD)患者甲状腺细胞凋亡的影响.方法 取5例GD患者甲状腺组织,采用原代细胞培养技术、细胞增殖实验和流式细胞术检测IFN-γ对甲状腺细胞增殖活力、细胞凋亡率以及对Fas蛋白表达率的影响.应用ELISA检测IFN-γ刺激甲状腺细胞后培养上清液中sFas含量的变化.结果 与未加IFN-γ处理的对照组相比,经IFN-γ处理的甲状腺细胞增殖活力明显降低(P＜0.05),但甲状腺细胞的凋亡率、Fas蛋白表达率以及培养上清液中sFas含量明显增加(P＜0.05);IFN-γ诱导的凋亡率与F&s蛋白表达率的相关性有统计学意义(P＜0.05).结论 IFN-γ可以通过调节Fas的表达影响甲状腺细胞凋亡的发生,提示IFN-γ可能通过调节凋亡参与GD的病理生理过程.     

自身免疫性甲状腺病患者血清免疫球蛋白及甲状腺自身抗体、ANA、抗ENA抗体变化及临床意义

目的 探讨自身免疫性甲状腺病(AITD)患者血清免疫球蛋白及甲状腺自身抗体、抗核抗体(ANA)、抗可溶性抗原(ENA)抗体水平变化及临床意义.方法 选取2017年1月1日至2020年8月1日我院收治的110例AITD患者作为研究对象,包括桥本甲状腺炎(HT)和毒性弥漫性甲状腺肿(GD),其中HT组共62例,GD组共48例,另外选取同期60例健康体检者作为对照组,采集所有受检者静脉血,应用免疫学方法分别检测其血清免疫球蛋白以及甲状腺自身抗体、ANA、抗ENA抗体水平.结果 3组自身免疫球蛋白IgM、IgG和IgA水平比较差异均具有统计学意义(P＜0.05),其中HT组患者IgM和IgG水平显著高于对照组(P＜0.05),GD组患者IgG和IgA水平显著高于对照组(P＜0.05),且HT组IgM和IgG水平显著高于GD组,IgA水平显著高于GD组(P＜0.05);3组TPOAb和TGAb水平比较差异均具有统计学意义(P＜0.05),其中HT组明显高于GD组和对照组,GD组明显高于对照组(P＜0.05);在AITD患者中ANA和抗双链DNA (ds-DN A)抗体阳性率显著高于对照组(P＜0.05),抗U1-RNP抗体阳性率比较差异无统计学意义(P＞0.05);而在所有受检者中均未检出ENA谱中其他抗体.结论 血清免疫球蛋白和血清甲状腺自身抗体检测对AITD的临床鉴别诊断具有一定积极的指导意义;ANA与抗ENA抗体在AITD中并无特异性,关于其确切病理机制和意义有待进一步深入研究.     

Graves病和桥本甲状腺炎患者外周血T细胞表面及血清中Fas/FasL的表达特点

目的探讨Graves病(GD)和桥本甲状腺炎(HT)患者外周血中Fas+、FasL+细胞占总T淋巴细胞的比例(Fas%、FasL%)以及血清中sFas、sFasL、TGAb、TPOAb等指标的变化及意义。方法选择GD患者36例、HT患者32例、对照组20例。用流式细胞术检测外周血T细胞表面Fa(sCD95)、FasL(CD178)的表达特点,用双抗体夹心法(ELISA)测定血清中可溶性Fas及FasL的含量,用化学发光法测定相关抗体TGAb、TPOAb的含量。结果 GD及HT患者外周血Fas%均高于对照组(P<0.05),且以HT组更为显著,而各组均未检测到FasL的表达;GD及HT患者血清中sFas含量均高于对照组,尤以GD组显著;各组间均可检测到sFasL,但差异无统计学意义(P>0.05)。结论 Fas及其配体介导的凋亡在GD和HT的自身免疫反应过程中起着重要的作用。流式细胞术的应用,可为探讨AITD的发病机制提供新的方法。     

子宫球蛋白相关蛋白1与桥本甲状腺炎的相关性

目的 探讨子宫球蛋白相关蛋白1(UGRP1)与桥本甲状腺炎(HT)的相关性。方法 通过免疫组化方法检测正常人、Graves病(GD)和桥本甲状腺炎患者甲状腺组织UGRP1的表达。选取60例自身免疫性甲状腺病(AITD)患者,其中HT患者30例为HT组,GD患者30例为GD组,选取无AITD的健康人28例为对照组,分别比较3组间临床一般资料、甲状腺相关指标、血清UGRP1水平。采用Pearson及Spearman相关性分析血清UGRP1水平与甲状腺相关指标的关系。结果 HT患者甲状腺组织UGRP1表达阳性,正常人、GD患者甲状腺组织UGRP1表达阴性。3组间临床一般资料差异无统计学意义(P>0.05)。3组间TT₃、TT₄、TSH、TPOAb、ATG、甲状腺体积水平比较,差异有统计学意义(P<0.05)。HT组血清UGRP1水平为1.39(1.06,1.85)ng/mL,高于GD组0.77(0.43,1.72)ng/mL及对照组0.60(0.29,1.03)ng/mL,差异有统计学意义(P<0.05)。Spearman相关分析...     

桥本甲状腺炎中细胞凋亡与增殖关系的实验研究

目的探讨桥本甲状腺炎(HT)中Fas、FasL与增殖细胞核抗原(PCNA)的关系.方法以非毒性甲状腺肿(NTG)的标本为对照,采用TUNEL及免疫组织化学双标记法,分别检测16例HT患者甲状腺标本中细胞凋亡水平及PCNA、Fas、FasL的表达及分布.结果凋亡率(AR)在NTG组为(0.9±0.64)%,HT组为(33.92±14.69)%;增殖率(PR)分别为(4.23±2.36)%和(8.45±3.61)%.AR、PR在HT组均显著高于NTG组(P＜0.01).HT中部分甲状腺滤泡细胞PCNA与Fas、FasL共表达;浸润淋巴细胞PCNA高表达,Fas/FasL弱或无表达.结论HT中甲状腺滤泡细胞凋亡与增殖活动均活跃,凋亡水平增高更为显著;浸润淋巴细胞增殖活跃,凋亡少见.其凋亡和增殖的失平衡可能是HT病理改变的主要机制之一.     

桥本甲状腺炎甲状腺组织细胞凋亡与凋亡基因Bc1-2、Bax表达

为了探讨桥本甲状腺炎(HT)甲状腺组织细胞凋亡的变化、凋亡基因Bc1-2、Bax的表达及其与HT发病机制的关系, 采用TUNEL法检测41例HT、10例正常甲状腺组织细胞凋亡状况, 同时采用免疫组化S-P法检测Bc1-2、Bax表达及分布, 应用Mias99图像分析系统对免疫组化结果进行定量分析.结果表明: HT组甲状腺细胞凋亡率及甲状腺组织Bc1-2、Bax阳性颗粒面积、平均光密度和积分光密度均显著高于正常组(P＜0.05-0.001);HT甲状腺滤泡区Bc1-2阳性颗粒面积和积分光密度与Bax的比值明显低于正常甲状腺的比值, 其组织结构破坏较重区域中小滤泡及失去正常滤泡结构的腺上皮Bax表达高于其它腺上皮, 滤泡结构较完整的区域Bc1-2表达较高, 提示甲状腺细胞凋亡增高是HT甲状腺滤泡结构破坏和功能异常的重要机制之一; 凋亡基因Bc1-2、Bax表达的失衡与HT时甲状腺细胞凋亡增高密切相关.     

自身免疫甲状腺病患者血清中IL-12和IL-18水平的分析

目的:提供自身免疫甲状腺病(AITD)患者体内Th1/Th2平衡紊乱的依据。方法:应用酶联免疫吸附法(ELISA)测定27例Graves病(GD)、24例甲状腺功能正常的桥本甲状腺炎(HT)、25例甲状腺功能低下的HT患者及20例正常对照者血清中IL12和IL18的浓度,并检测GD患者的甲状腺刺激性抗体。结果:GD患者与甲状腺功能正常的HT患者血中IL12、IL18水平无明显差异,但均高于正常对照者的相应水平。甲状腺功能低下的HT患者血中IL12和IL18的水平与正常对照者无差异。在GD和甲功正常的HT,IL18与IL12呈明显正相关。在GD,IL12和IL18均与其甲状腺刺激性抗体(TSAb)活性呈正相关。在甲状腺功能正常的HT还存在IL12和IL18二者与甲状腺球蛋白抗体(TgAb)的显著性正相关。结论:提示Th1型细胞在GD和HT两种AITD的发病中均起重要作用。通过抑制Th2型免疫反应,促进向Th1型的转变来治疗GD时,有可能导致病情恶化。     

Identification of apoptotic proteins in thyroid gland from patients with Graves' disease and Hashimoto's thyroiditis

Apoptosis, i.e. natural programmed cell death, is a physiological phenomenon indispensable for normal functioning of the organism. The signal to apoptosis can be started practically in any cell. Disturbances in the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of the study was to assess the expression of Fas/FasL and caspase eight in the tissues of the thyroid gland in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). The analysis of Fas/FasL expression was performed by western blot and immunohistochemical investigation with DAB-visualization and Mayer's hematoxylin staining. Caspase-8 expression in thyroid follicular cells was assayed by western blot method. Identification of the proapoptotic proteins FasL and Fas exhibited their pronounced expression in the thyroid tissue in GD patients (++; ++) and HT (+++; +++) as compared to the NTNG group (0/+; 0/+). Among the study groups, the expression of caspase-8 was revealed in band 55 kDa from patients with autoimmune thyroid diseases. In GD patients, the percentage of thyrocytes with FasL expression correlated positively with TRAb (R = 0.58, p < 0.02). However, no such correlations were noted in HT or non-toxic multinodular goiter. There were no significant correlations between thyroid hormones and the percentage of thyrocytes with Fas and FasL expression. In conclusion, our findings suggest that the changes in the expression of apoptotic molecules on the surface of T lymphocytes and thyroid follicular cells in patients with autoimmune thyroid disorders reflect their substantial involvement in the pathogenesis of GD and HT. In addition, analysis of Fas/FasL and caspase-8 expression in thyroid tissue may indicate the disease activity and immunological phenotype.     

Identification of apoptotic proteins in thyroid gland from patients with Graves' disease and Hashimoto's thyroiditis

Apoptosis, i.e. natural programmed cell death, is a physiological phenomenon indispensable for normal functioning of the organism. The signal to apoptosis can be started practically in any cell. Disturbances in the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders.

The aim of the study was to assess the expression of Fas/FasL and caspase eight in the tissues of the thyroid gland in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). The analysis of Fas/FasL expression was performed by western blot and immunohistochemical investigation with DAB-visualization and Mayer's hematoxylin staining. Caspase-8 expression in thyroid follicular cells was assayed by western blot method.

Identification of the proapoptotic proteins FasL and Fas exhibited their pronounced expression in the thyroid tissue in GD patients (++; ++) and HT (+++; +++) as compared to the NTNG group (0/+; 0/+). Among the study groups, the expression of caspase-8 was revealed in band 55 kDa from patients with autoimmune thyroid diseases.

In GD patients, the percentage of thyrocytes with FasL expression correlated positively with TRAb (R = 0.58, p < 0.02). However, no such correlations were noted in HT or non-toxic multinodular goiter. There were no significant correlations between thyroid hormones and the percentage of thyrocytes with Fas and FasL expression.

In conclusion, our findings suggest that the changes in the expression of apoptotic molecules on the surface of T lymphocytes and thyroid follicular cells in patients with autoimmune thyroid disorders reflect their substantial involvement in the pathogenesis of GD and HT. In addition, analysis of Fas/FasL and caspase-8 expression in thyroid tissue may indicate the disease activity and immunological phenotype.     

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

凋亡相关因子参与自身免疫性甲状腺疾病发病的研究

目的:了解细胞凋亡相关蛋白Fas,FasL和Bcl-2表达在自身免疫性甲状腺疾病发病机制及病理变化中的作用及意义。方法:采用免疫组织化学方法,检测20例桥本甲状腺炎,20例Graves病以及20例甲状腺腺瘤(作为对照组)患者甲状腺标本中Fas、FasL和Bcl-2表达及分布。结果:Fas在所有的标本中表达,主要分布于甲状腺滤泡细胞表面和细胞质上。除3例甲状腺瘤标本外,其余均表达FasL。Bcl-2表达于15例桥本甲状腺炎、19例Graves病以及17例甲状腺瘤滤泡细胞上。在甲状腺瘤滤泡细胞上表达中等强度Fas,很少或是没有表达FasL。在桥本甲状腺炎中Fas和FasL免疫染色强阳性甲状腺滤泡细胞多分布于浸润淋巴滤泡附近,浸润淋巴细胞中Fas、FasL免疫染色相对较弱。在Graves病中,Fas表达强度与桥本甲状腺炎类似,但FasL表达却更弱。在Graves病和甲状腺瘤组织中,Bcl-2表达两者类似。但在桥本甲状腺炎组织中,分布于浸润淋巴细胞附近的甲状腺滤泡细胞以及生发中心的淋巴细胞上,Bcl-2表达很弱。结论:Fas、FasL和Bcl-2表达在桥本甲状腺炎和Graves病中相似。FasL高表达和Bcl-2低表达可能引起桥本甲状腺炎滤泡细胞凋亡。进一步证明3种凋亡相关因子在自身免疫性甲状腺疾病发病机制中的作用。在桥本甲状腺炎中,滤泡细胞凋亡并非由浸润淋巴细胞其FasL发挥作用直接杀伤,但是它们能分泌细胞因子促进滤泡细胞自身Fas、FasL表达,从而导致滤泡细胞凋亡。     

Analysis of the expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disorders

To investigate the expression of apoptosis-related protein (Fas, FasL, and Bcl-2) in the pathogenesis of autoimmune thyroid disorders (ATDs), immunohistochemical staining was performed on 20 Hashimoto‘s thyroiditis (HT), 20 Graves‘ disease (GD), and 20 thyroid follicular adenoma (TFA, as control). All the cases expressed Fas, mainly on the cell surface and cytoplasm. FasL was found in 17 cases of the TFA. Bcl-2 was detected in 15 cases of HT, 19 of GD and 17 of TFA. In T FA, a moderate Fas expression and a minimal or no FasL expression was detected on follicular cells. In HT, the follicles adjacent to infiltrating lymphocytes showed increased levels of Fas and FasL expression. A weaker staining of Fas and FasL was exhibited on infiltrating lymphocytes than on thyrocytes. In a comparison of GD with HT, thyrocytes and lymphocytes showed similar Fas staining, but for FasL the staining was rather weaker in HT. The expression of Bcl-2 was nearly identical in GD and TFA, but much weaker on the follicular cells in vicinity of lymphocytes and on the lymphocytes located in germinal centers of HT tissues. The expression of Fas, FasL, Bcl-2 in Hashimoto‘s thyroiditis and Graves‘ disease were almost same. FasL strong expression and Bcl-2 weak expression on the follicles in HT may induce apoptosis. These results provided evidence for expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disease. The lymphocytes seem not to be directly engaged in the process v/a their own FasL, but they may provide some cytokines that, in turn, upregulate Fas and/or FasL expression to induce apoptosis.     

测定血清TGAb和TPOAb在自身免疫性甲状腺疾病中的临床价值

为探讨自身免疫性甲状腺疾病(AITD)患者血清TGAb和TPOAb浓度及临床价值,用RIA测定175例AITD组患者、64例非AITD组患者和57名对照组血清TGAb和TPOAb浓度.结果表明,AITD组中GD、HT患者血清TGAb和TPOAb浓度显著高于对照组(P<0.01),而非AITD组与对照组比较无显著性差异(P>0.05).本文认为检测TGAb和TPOAb有助于了解AITD的发病机制,对AITD的诊治及预后判断具有一定的临床价值.     

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture of IgG and C3 fragments. Furthermore, the binding of Tg to B cells in preparations of normal blood cells was higher in HT serum than in serum from controls and correlated positively with the serum anti-Tg activity, as did the B and CD4+ T cell proliferation. Disruption of the three-dimensional structure of Tg by boiling reduced the proliferative responses. The data indicate that anti-Tg antibodies associated with AITD facilitate the formation of complement-activating Tg/anti-Tg complexes, binding of IC to B cells, and the subsequent proliferation of B and T cell subsets. This represents a novel mechanism for the maintenance of autoimmune processes in AITD and links autoreactive T cell responses with the presence of autoantibodies.     

血清抗甲状腺球蛋白抗体和抗甲状腺过氧化物酶抗体检测在甲状腺疾病诊断及甲亢预后判断中的价值

采用放免法检测甲状豚疾病患者抗甲状腺球蛋白抗体（ＴＧＡｂ）和抗甲状腺过氧化物酶抗体（ＴＰＯＡｂ）、并对部分Ｇｒａｖｅｓ病患者停药后随诊一年的结果进行分析。结果显示：（１）自身免疫性甲状腺疾病患者ＴＧＡｂ和ＴＰＯＡｂ活性及阳性率明显高于非ＡＩＴＤ，尤以桥本甲状腺炎为然。（２）ＧＤ治疗前及停药时ＴＧＡｂ和ＴＰＯＡｂ均阴性者与均阳性者停药一年内的复发率分别为０．５８３和０．２３１。（３）ＴＧＡｂ和ＴＰＯＡｂ均阴性，而停药时甲状腺刺激抗体（ＴＳＡｂ）阳性者，停药时ＧＤ复发的机率最大（０．９０９），提示ＴＧＡｂ和ＴＰＯＡｂ检测在ＡＩＴＤ诊断，鉴别诊断以及ＧＤ预后判断中具有重要的临床意义。