自身免疫性甲状腺疾病甲状腺组织中bcl-2家族蛋白的表达

目的研究凋亡相关基因bcl鄄2家族蛋白bcl鄄2、mcl鄄1、bcl鄄XL和bax在自身免疫性甲状腺疾病(AITD)甲状腺组织中的表达特征及与AITD发病机制之间的内在联系。方法以甲状腺腺瘤旁正常甲状腺组织为对照(C组,20例),采用免疫组织化学ElivisionTM二步染色法检测凋亡相关蛋白bcl鄄2、mcl鄄1、bcl鄄XL和bax在桥本甲状腺炎(HT组,33例)和Graves病(GD组,28例)患者甲状腺组织中的表达与分布。结果bcl鄄2蛋白表达强度GD组>C组>HT组(P<0.01);mcl鄄1蛋白表达强度GD组>C组>HT组(P<0.01);bcl鄄XL蛋白表达强度HT组和GD组强于C组(P<0.01),但HT组和GD组间差异无统计学意义(P>0.05);bax蛋白的表达强度HT组>GD组和C组(P<0.01),但GD组和C组间差异无统计学意义(P>0.05);HT组中,在淋巴细胞浸润区域附近的甲状腺滤泡上皮细胞(TEC)bcl鄄2表达弱,bax和mcl鄄1表达强;远离淋巴细胞浸润区域的TECbcl鄄2表达强,bax和mcl鄄1表达弱(P<0.05)。结论(1)抗凋亡bcl鄄2和mcl鄄1蛋白在HT中表达的减弱以及在GD中表达的增强对于HT甲状腺滤泡细胞凋亡的增加和GD甲状腺滤泡细胞的增殖可能起一定作用;(2)bax蛋白在HT中表达增强所起的促凋亡的作用对疾病的发生发展起一定作用;(3)bcl鄄2与bax表达强度的比值对于凋亡的调控起重要作用;(4)bcl鄄2家族蛋白bc     

自身免疫性甲状腺疾病甲状腺组织中凋亡调控蛋白Fas/FasL、Bcl-2/Bax的表达及意义

目的探讨凋亡调控蛋白Fas/FasL、Bcl-2/Bax在Graves病(GD)和桥本氏病(HT)中的表达及意义。方法取外科手术切取的GD及HT病人甲状腺组织标本,颈部手术时取正常甲状腺组织作为对照;采用免疫组织化学方法检测甲状腺标本Fas/FasL、Bcl-2/Bax的表达和分布状况。结果(1)Fas/FasL在GD和HT甲状腺滤泡上皮细胞表达均较正常对照组增高(P<0.01)。(2)在GD甲状腺滤泡上皮细胞Bcl-2及Bax表达明显高于HT组及正常对照组,以Bcl-2表达强度显著高于同组Bax(P<0.01),而在HT,Bcl-2表达强度与正常对照组比较差异无统计学意义。(3)GD与HT相比较,GD甲状腺滤泡细胞及浸润淋巴细胞Fas,Bcl-2抗原表达均强于HT,但其浸润淋巴细胞表达强度明显低于同组甲状腺滤泡细胞(均P<0.05)。结论Fas/FasL高表达和Bcl-2的低表达可能引起HT甲状腺滤泡上皮细胞的凋亡。在GD,Fas、Bax诱导细胞凋亡的作用可能被高表达Bcl-2对抗,从而致甲状腺体积增大、功能亢进。此外,Bcl-2与Bax表达强度的比值对于凋亡的调控有重要影响作用。     

自身免疫性甲状腺疾病患者甲状腺内Th1/Th2细胞失衡研究

目的　探讨甲状腺中Th1/Th2 细胞失衡在自身免疫性甲状腺疾病 (AITD)发病中的作用。方法　选取 13例Graves病 (GD)患者、12例桥本甲状腺炎 (HT)患者 ,并以 8例非毒性结节性甲状腺肿患者作为对照进行研究。采用免疫组化染色方法检测这些患者甲状腺内单个核细胞 (ITMC)的γ 干扰素(IFN γ)和白介素 4(IL 4)细胞因子抗原表达 (分别代表Th1,Th2 分泌的细胞因子 ) ,并与外周血甲状腺刺激性抗体 (TSAb)、甲状腺球蛋白抗体 (TGAb)、甲状腺微粒体抗体 (TMAb)等免疫学指标进行相关分析。结果　 ( 1)GD、HT患者ITMC的IL 4、IFN γ阳性表达明显高于对照组 (均P <0 .0 1) ;GD患者ITMC的IL 4阳性表达明显高于IFN γ阳性表达 ;而HT患者ITMC的IFN γ阳性表达则明显高于IL 4阳性表达(均P <0 .0 5 ) ;( 2 )GD患者ITMC的IL 4阳性表达与TSAb显著正相关 (r =0 .67,P <0 .0 1) ,ITMC的IFN γ阳性表达与TSAb无相关性 ;HT患者ITMC的IFN γ阳性表达与TGAb、TMAb均呈显著正相关(分别为r =0 .65 ,r =0 .5 9,均P <0 .0 5 ) ,但ITMC的IL 4阳性表达与TGAb、TMAb均无相关性。结论　GD患者Th1/Th2 细胞平衡失衡偏向以Th2 占优势的免疫应答 ,而HT患者Th1/Th2 平衡失衡偏向以Th1占优势的免疫应答。     

缝隙连接蛋白43、26在自身免疫性甲状腺疾病患者甲状腺上皮细胞中的表达

目的 探讨缝隙连接蛋白43、26(Cx43、Cx26)在自身免疫性甲状腺疾病(AITD)患者甲状腺上皮细胞中的表达,探索其在AITD的发病机制、转归及预后中所起的作用.方法 采用二步法免疫组织化学技术(S-P法)测定甲状腺腺瘤旁正常甲状腺组织(对照组,16例)、Graves病(GD组,30例)和桥本甲状腺炎(HT组,30例)患者甲状腺上皮细胞中Cx43、Cx26的分布及表达情况.结果 ①对照、GD和HT组甲状腺上皮细胞均有Cx43、Cx26的表达.既定位于细胞质中,又定位于细胞膜上.②对照、GD和HT组甲状腺上皮细胞Cx43表达阳性率分别为75.00%(12/16)、100.00%(30/30)和33.33%(10/30),Cx26表达阳性率分别为68.75%(11/16)、100.00%(30/30)和20.00%(6/30).Cx43、Cx26在GD组的表达强度均明显高于对照组(Z值分别为4.782、5.310,P均<0.017),在HT组的表达强度均明显低于对照组(Z值分别为2.703、3.123,P均<0.017).结论 Cx43、Cx26可在人甲状腺上皮细胞中表达;Cx43、Cx26在AITD的表达强度存在异质性,这种缝隙连接蛋白表达的异质性可能与AITD的发生、发展及预后有关.     

不同类型甲状腺疾病患者可溶性细胞间黏附分子-1变化及临床意义

目的探讨不同类型甲状腺疾病患者可溶性细胞间黏附分子-1(sICAM-1)变化及临床意义。方法选择初诊甲状腺疾病患者137例,其中Graves病(GD)69例(GD组)[伴Graves眼病(GO)20例],桥本甲状腺炎(HT)35例(HT组),单纯性甲状腺肿(SG)20例(SG组),非毒性结节性甲状腺肿(NTNG)13例(NTNG组)。另选健康查体者277例作为对照组。采用125I-sICAM-1 RIA方法检测各组血清sICAM-1,化学发光法检测FT3、FT4、sTSH,放免法检测甲状腺球蛋白抗体(TGAb)、甲状腺微粒体抗体(TMAb),ELISA法检测促甲状腺素受体抗体(TRAb)、甲状腺刺激免疫球蛋白(TSI);采用Spearman等级相关分析,分析sICAM-1与甲状腺功能及甲状腺自身抗体指标的相关性。结果与对照组比较,GD组、HT组sICAM-1明显升高(P均<0.01),GD组与HT组、SG组与NTNG组比较,P均>0.05;GO患者的sICAM-1明显高于非GO患者(P<0.05)。GD组sICAM-1与FT3、FT4呈正相关(r分别为0.467、0.441,P均<0.05),与TRAb、TSI、TGAb、TMAb无明显相关性(P均>0.05)。结论血清sI-CAM-1可作为免疫指标之一,辅助诊断GD、HT等甲状腺疾病。     

Th1/Th2细胞平衡偏移与自身免疫性甲状腺疾病的相关性研究

目的 明确白细胞介素-2(IL-2)、IL-5与自身免疫性甲状腺疾病(AITD)发生的相关性,探讨Th1/Th2细胞平衡在自身免疫性甲状腺疾病发病中的作用.方法 选取2007年8月至2008年5月南昌大学第三附属医院内分泌科45例Graves病(GD)、30例桥本甲状腺炎(HT)患者,同时选取20名健康体检者作为对照组.采用酶联免疫吸附试验法(ELISA)检测患者和对照组血清中Th1型细胞因子IL-2扣Th2型细胞因子IL-5并进行统计学分析.结果 (1)GD患者血清中IL-5明显高于HT组和对照组(P<0.05),而IL-2与对照组相比差异无统计学意义(P>0.05).(2)HT患者血清IL-2明显高于GD组和对照组(P<0.05),而IL-5与对照组相比差异无统计学意义(P>0.05).(3)HT患者Th1型细胞因子IL-2与甲状腺球蛋白抗体(TCAb)和甲状腺微粒体抗体(TMAb)呈显著正相关(r=0.517,P<0.05;r=0.475,P<0.01).结论 Th1/Th2细胞免疫平衡在HT患者中以Th1细胞免疫占优势为主,在GD患者中以Th2细胞免疫占优势为主.     

甲状腺特异性转录因子对甲状腺功能的调控

甲状腺特异性转录因子是调控甲状腺功能的小分子蛋白,包括甲状腺转录因子(TTF)-1、双链复合蛋白(Pax)-8和TTF-2。生理状态下,促甲状腺激素、胰岛素和胰岛素样生长因子-1通过对TTF-1、TTF-2和Pax-8的表达及活性的调节,上调钠碘同向转运体、甲状腺过氧化物酶、甲状腺球蛋白、促甲状腺激素受体的表达,调节甲状腺功能。Pax-8和TTF-1也可使主要组织相容性复合物Ⅰ表达下调,使甲状腺可以耐受自身激素合成过程中增加的基因产物。TTF-1也可影响人类各组织中5′脱碘酶2型以及甲状腺滤泡旁细胞(C细胞)和甲状旁腺细胞中钙离子转运相关基因的表达。     

甲状腺转移因子—1与肺癌

甲状腺转移因子-1(TTF-1)是近年来发现的一个新的肿瘤标志物，主要表达于甲状腺肿瘤及肺肿瘤。近年来的研究表明，TTF-1在肺肿瘤特别是肺腺癌的表达具有高度的敏感性和特异性。检测TTF-1可有效地提高肺癌的诊断率，特别是能够鉴别恶性肿瘤的原发部位，也可用于肺癌的预后判断。     

Graves病患者甲状腺组织内IL-6及gp130表达增强

对8例Graves病(GD)甲状腺组织内白细胞介素6(IL-6)及其受体(gp80、gp130)表达进行研究,发现GD组甲状腺组织内IL-6mRNA、gp130mRNA表达增高;GD中甲状腺滤泡细胞和淋巴细胞胞浆IL-6表达水平显著升高.提示GD患者甲状腺内IL-6/gp130信号通路的激活可能参与GD的发病.     

CD54、CD80、HLA-DR在Graves病和桥本甲状腺炎甲状腺滤泡上皮中的表达

目的观察正常人、Graves病（GD）、桥本甲状腺炎（HT）、甲状腺组织CD54、CD80、HLA—DR的表达。方法免疫组化SP法测定上述各组甲状腺组织CD54、CD80、HLA—DR的表达水平。结果正常甲状腺组织甲状腺滤泡上皮细胞（TEC）几乎不表达CD54和HLA—DR，而GD组和HT组有较高水平的CD54和HLA-DR表达，以HT表达水平最高，3组间比较差异有统计学意义（均P〈0．01）；HT患者甲状腺CD80表达高于GD组和正常人，差异有统计学意义（均P〈0．01），GD组甲状腺CD80表达很低，与正常人相比差异没有统计学意义。结论CD54和HLA-DR在GD和HT的发病机制中起重要作用，CD80在HT的发病中起重要作用。     

Human thyroid phenol sulfotransferase enzymes 1A1 and 1A3: activities in normal and diseased thyroid glands, and inhibition by thyroid hormones and phytoestrogens

Sulfation by sulfotransferase enzymes (SULTs) is an important pathway for the metabolism of thyroid hormones and phytoestrogens. Intrathyroidal SULTs may contribute to the processing of thyroid hormones for the reutilization of iodide. SULT1A1 and SULT1A3 activities were identified in normal and diseased human thyroid glands. Biochemical properties that included apparent K(m) values, thermal stabilities, and responses to inhibitors were characterized in a normal human thyroid high speed supernatant pool. Apparent K(m) values for SULT1A1 and SULT1A3 activities with the model substrates p-nitrophenol and dopamine were 0.58 +/- 0.04 and 11.3 +/- 1.3 microm, respectively. Activities of SULT1A1 and SULT1A3 determined in individual normal thyroid (n = 35), nodular goiter (n = 26), and autoimmune thyroid disease (n = 25) glands were 0.34 +/- 0.06, 0.52 +/- 0.09, and 0.82 +/- 0.19 U/mg protein for SULT1A1, respectively, and 0.22 +/- 0.04, 0.21 +/- 0.04, and 0.48 +/- 0.11 U/mg protein for SULT1A3, respectively. Both SULT activities in autoimmune thyroid disease glands were significantly higher than those in normal thyroids. Only 3,3'-diiodothyronine (3,3'-T(2)) and the phytoestrogen daidzein served as substrates for the normal thyroid SULT activities, yet each thyroid hormone and phytoestrogen tested were found to inhibit thyroid SULT1A1 and SULT1A3 activities. The preference of thyroid gland SULT activities for 3,3'-T(2) suggests that sulfation may enhance degradation of intrathyroidal 3,3'-T(2) for iodide reutilization. Inhibition of these SULT activities by the exogenous phytoestrogens daidzein and genistein, with a potential decrease in iodide reutilization, presents another mechanism through which these compounds may adversely affect human thyroid function.     

Characterisation of DEHAL1 expression in thyroid pathologies

Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide in the human thyroid. The aim of the present study was (I) to investigate whether DEHAL1 expression is different in differentially functioning thyroid pathologies and (II) to evaluate DEHAL1 as a possible marker of thyroid cell differentiation. DESIGN AND METHODS: Real-time PCR for DEHAL1 and its isoform DEHAL1B was performed in a series of 105 thyroid specimens, including toxic thyroid nodules (TTN), Graves' disease (GD) thyroids, benign cold thyroid nodules (CTN), normal thyroid tissues and thyroid cancers (follicular thyroid carcinomas (FTC), papillary thyroid carcinomas (PTC), partially differentiated thyroid cancers (PDTC) and anaplastic thyroid carcinomas (ATC)). In addition, DEHAL1 protein expression was studied by immunohistochemistry in 163 benign and malignant thyroid pathologies and normal thyroids. RESULTS: (I) The highest DEHAL1 mRNA levels were found in GD thyroids, while downregulation of DEHAL1 and DEHAL1B mRNA occurred in PTC and ATC (P<0.001 and <0.05 respectively); (II) DEHAL1 protein was overexpressed in TTNs and GD thyroids with predominant apical staining in all samples; (III) a weaker and patchy staining pattern was found in CTNs and normal thyroids; (IV) in differentiated thyroid cancers (FTC and PTC), a diffuse cytoplasmic DEHAL1 expression was found; and (V) in PDTC and ATC, DEHAL1 expression was faint or absent. CONCLUSION: Upregulation of DEHAL1 protein expression and sublocalisation of DEHAL1 at the apical cell pole in TTNs and GD thyroids is consistent with increased thyroid hormone turnover during thyrotoxicosis. Diffuse cytoplasmatic localisation or downregulation of DEHAL1 expression in thyroid cancers suggests alteration or loss of DEHAL1 function during thyroid cell dedifferentiation.     

甲状腺激素反应蛋白1的原核生物表达

目的 应用基因工程技术表达甲状腺激素反应蛋白1(TRP-1)。方法 应用RT-PCR方法，从新生大鼠脑组织RNA中扩增编码TRP-1的cDNA片段，构建表达型重组质粒，经DNA序列分析确证后，在大肠杆菌中表达，以Western印迹来初步鉴定是否表达了目的融合蛋白，用亲和层析纯化融合目的蛋白，并以SDS-PAGE电泳测定其相对分子质量。结果 所获特异PCR产物正确地重组入Pinpoint Xa-1表达载体中。Western印迹表明经异丙基硫代半乳糖苷诱导的原核细胞表达产生了目的融合蛋白，亲和层析得到了纯度较高、相对分子质量约23400的融合目的蛋白。结论 应用原核细胞表达体系成功地表达了TRP-1融合蛋白，为进一步研究其在脑发育中的所起的作用以及该蛋白的其它功能提供了可能。     

sFlt-1 gene therapy of follicular thyroid carcinoma

Tumor progression largely depends on blood supply and neovessel formation, and angiogenesis is emerging as a promising target for cancer therapy. Vascular endothelial growth factor (VEGF), a major proangiogenic molecule, stimulates angiogenesis via promoting endothelial proliferation, survival and migration. VEGF has been found to be up-regulated in various types of tumors and to be associated with tumor progression and poor prognosis. Inhibition of VEGF or its signaling pathway has been shown to suppress tumor angiogenesis and tumor growth. In the present study, we tested the antiangiogenic and antitumor effects of soluble VEGF receptor-1 [soluble Flt (sFlt)-1] on the growth of follicular thyroid carcinoma (FTC). We constructed a 293 embryonic kidney cell line (293-Flt1-3d) that expresses sFlt-1, which is composed of the first three extracellular domains of Flt-1. The 293-Flt1-3d cells inhibited the in vitro growth of human umbilical vein endothelial cells in a paracrine manner. The in vivo antitumor and antiangiogenic activities of the 293-Flt1-3d cells were tested. When 293-Flt1-3d cells were inoculated at a site remote to the FTC-133 tumor transplant, the growth of FTC-133 tumors were inhibited by 70.37%, as compared with the control treatment with 293 cells expressing control gene LacZ. Immunohistochemical analysis of microvessel densities in treated tumors demonstrated that 293-Flt1-3d cells robustly suppressed intratumoral angiogenesis. Our data suggest that a mammalian cell-mediated approach could effectively deliver sFlt-1 gene therapy and inhibit tumor angiogenesis and tumor growth.     

NCoR1 regulates thyroid hormone receptor isoform-dependent adipogenesis

We previously showed that two thyroid hormone receptor (TR) isoforms--TRα1 and TRβ1--differentially regulate thyroid hormone (triiodothyroxine, T(3))-stimulated adipogenesis in vivo. This study aims to understand the role of the nuclear receptor corepressor, NCoR1, in TR isoform-dependent adipogenesis. We found that T(3)-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR1 protein levels. In 3T3-L1 cells stably expressing a mutated TRα1, PV (L1-α1PV cells), the T(3)-stimulated adipogenesis was more strongly inhibited than that in 3T3-L1 cells stably expressing an identical mutation in TRβ1 (L1-β1PV cells). The stronger inhibition of adipogenesis in L1-α1PV cells was associated with a higher NCoR1 protein level. These results indicate that the degree of loss of NCoR1 correlates with the extent of adipogenesis. siRNA knockdown of NCoR1 promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-α1PV and L1-β1PV cells, indicating that NCoR1 plays an essential role in TR isoform-dependent adipogenesis. An ubiquitin ligase, mSiah2, that targets NCoR1 for proteasome degradation was upregulated on day 1 before the onset of progressive loss of NCoR1. NCoR1 was found to associate with mSiah2 and with TR, TRα1PV, or TRβ1PV, but a stronger interaction of NCoR1 with TRα1PV than with TRβ1PV was detected. Furthermore, TRα1PV-NCoR1 complex was more avidly recruited than TRβ1PV-NCoR1 to the promoter of the C/ebpα gene, leading to more inhibition in its expression. These results indicate that differential interaction of NCoR1 with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis and that aberrant interaction of NCoR1 with TR could underlie the pathogenesis of lipid disorders in hypothyroidism.     

Interleukin-1 production and action in thyroid tissue

This study was undertaken to determine the effects of interleukin-1 (IL-1) on human thyroid epithelial cells (thyrocytes) and whether thyrocytes produce IL-1. The supernatants of cultured peripheral blood monocytes stimulated with lipopolysaccharide (LPS) increased [3H]thymidine incorporation into thyrocytes from normal subjects and patients with Grave's disease. The IL-1 levels of cultured supernatants of monocytes were measured by a thymocyte costimulation assay and a solid phase sandwich immunoenzymometric assay. The supernatants of monocyte cultures stimulated with LPS contained significant amounts of IL-1 bioactivity and IL-1 alpha and IL-1 beta immunoactivity. Recombinant IL-1 beta (rIL-1 beta) also stimulated [3H]thymidine incorporation into thyrocytes from normal subjects and patients with Graves' disease, and it increased the proportion of thyrocytes in the S phase of the cell cycle. Furthermore, thyrocytes stimulated with rIL-1 beta for 24 h produced significant amounts of prostaglandin E2. Indomethacin inhibited completely the rIL-1 beta-stimulated prostaglandin E2 production and increased markedly [3H]thymidine incorporation. IL-1-like activity also was detected in the cultured supernatants of lipopolysaccharide (LPS)-stimulated thyrocytes from Graves' and normal thyroid glands, but the amount of IL-1-like activity secreted by thyrocytes was significantly less than that secreted by circulating monocytes. The kinetics of the release of IL-1-like activity by thyrocytes were similar to those of its production by circulating monocytes. Pretreatment of thyrocytes with interferon-gamma failed to enhance the release of IL-1-like activity. Moreover, IL-1 alpha or IL-1 beta immunoreactivity could not be detected in the supernatants of LPS-stimulated thyrocytes, despite the presence of IL-1-like bioactivity. No IL-1 alpha mRNA was detected in unstimulated thyrocytes or thyrocytes stimulated with LPS and phorbol myristate acid. These findings demonstrate that thyrocytes produce an IL-1-like substance(s), but not IL-1, when stimulated by LPS. We conclude that IL-1 may regulate the proliferation of thyrocytes and that local production of IL-1 by infiltrating monocytes may contribute to the development of goiter in patients with autoimmune thyroid diseases.