CAJ | 학술논문

目的观察分析共刺激分子CD40在格雷夫斯病甲状腺组织中的表达及其免疫病理意义.方法选取2008年1月至2010年12月南京医科大学附属苏州医院和苏州大学附属第三人民医院收治的甲状腺手术患者甲状腺组织8例,其中3例为格雷夫斯病,5例为非毒性甲状腺肿.运用免疫组织化学染色技术检测CD40分子在甲状腺组织中的表达；运用原代培养技术体外培养甲状腺滤泡细胞(TFC),分别加入细胞因子γ干扰素(IFN-γ)(IFN-γ刺激组)、白细胞介素(IL)-6(IL-6刺激组)或肿瘤坏死因子-α(TNF-α)(TNF-α刺激组)刺激,设不加细胞因子组为无刺激组,培养3d后,用流式细胞术检测各组TFC表面CD40分子的表达；加入激发型CD40单抗5C11(IFN-γ+CD40组)或小鼠IgG抗体(IFN-γ+mIgG组)与IFN-γ刺激组TFC共培养3d后,用噻唑蓝(MTT)法检测TFC的体外增殖；放免法检测各组TFC培养上清中游离三碘甲腺原氨酸(FT3)、游离甲状腺素(FT4)和甲状腺球蛋白(Tg)的水平.结果 CD40分子表达于格雷夫斯病患者甲状腺组织中TFC表面,而在非毒性甲状腺肿TFC表面未见阳性表达.IFN-γ刺激组、IL-6刺激组、TNF-α刺激组原代培养TFC表面CD40分子的表达分别为86.4％±4.6％、90.0％±4.2％、87.3％ ±4.2％,均显著高于无刺激组的23.7％ ±7.3％(均P＜0.01).IFN-γ+CD40组TFC增殖能力明显高于IFN-γ+mIgG组(1.14±0.14比0.75 ±0.06,P＜0.01)；IFN-γ+CD40组TFC培养上清中分泌的FT3、FT4和Tg分别为(1.10±0.15)、(0.80 ±0.14) pmol/L和(30.23±1.60) μg/L,均显著高于IFN-γ+ mIgG组的(0.76 ±0.07)、(0.63 ±0.09) pmol/L和(21.37±3.22)μg/L(均P＜0.05).结论共刺激分子CD40异常表达于格雷夫斯病患者甲状腺组织,其介导的共刺激信号可能参与了格雷夫斯病的免疫病理机制.
목적 관찰분석공자격분자CD40재격뢰부사병갑상선조직중적표체급기면역병리의의.방법 선취2008년1월지2010년12월남경의과대학부속소주의원화소주대학부속제삼인민의원수치적갑상선수술환자갑상선조직8례,기중3례위격뢰부사병,5례위비독성갑상선종.운용면역조직화학염색기술검측CD40분자재갑상선조직중적표체；운용원대배양기술체외배양갑상선려포세포(TFC),분별가입세포인자γ간우소(IFN-γ)(IFN-γ자격조)、백세포개소(IL)-6(IL-6자격조)혹종류배사인자-α(TNF-α)(TNF-α자격조)자격,설불가세포인자조위무자격조,배양3d후,용류식세포술검측각조TFC표면CD40분자적표체；가입격발형CD40단항5C11(IFN-γ+CD40조)혹소서IgG항체(IFN-γ+mIgG조)여IFN-γ자격조TFC공배양3d후,용새서람(MTT)법검측TFC적체외증식；방면법검측각조TFC배양상청중유리삼전갑선원안산(FT3)、유리갑상선소(FT4)화갑상선구단백(Tg)적수평.결과 CD40분자표체우격뢰부사병환자갑상선조직중TFC표면,이재비독성갑상선종TFC표면미견양성표체.IFN-γ자격조、IL-6자격조、TNF-α자격조원대배양TFC표면CD40분자적표체분별위86.4％±4.6％、90.0％±4.2％、87.3％ ±4.2％,균현저고우무자격조적23.7％ ±7.3％(균P＜0.01).IFN-γ+CD40조TFC증식능력명현고우IFN-γ+mIgG조(1.14±0.14비0.75 ±0.06,P＜0.01)；IFN-γ+CD40조TFC배양상청중분비적FT3、FT4화Tg분별위(1.10±0.15)、(0.80 ±0.14) pmol/L화(30.23±1.60) μg/L,균현저고우IFN-γ+ mIgG조적(0.76 ±0.07)、(0.63 ±0.09) pmol/L화(21.37±3.22)μg/L(균P＜0.05).결론 공자격분자CD40이상표체우격뢰부사병환자갑상선조직,기개도적공자격신호가능삼여료격뢰부사병적면역병리궤제.
Objective To explore the expression of costimulatory molecule CD40 in thyroid tissue of Graves' disease patients and understand its immune pathogenetic significance.Methods From January 2008to December 2011,8 patients undergoing partial thyroidectomy for Graves' disease (n =3) or non-toxic goiter (n =5) at Affiliated Suzhou Hospital of Nanjing Medical University and Third Affiliated Hospital of Soochow University were enrolled.Using the method of immunohistochemistry,the expression of CD40 was detected in their thyroid tissues.Variation in CD40 expression on thyroid follicular (TFC) in primary cultures was analyzed in the absence (no stimulation group) or presence of inflammatory cytokines including interleukin interferon-γ (IFN-γ) (IFN-γ stimulation group),interferon-6(IL-6) (IL-6 stimulation group) and tumor necrosis factor (TNF)-α (TNF-α stimulation group) with flow cytometry.IFN-γ-stimulated TFC were cultured with agonist CD40 monoclonal antibody (5C11) (IFN-γ + CD40 group) or isotypic mouse IgG (mIgG) antibody (IFN-γ + mIgG group).And the proliferation of TFC was assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays for each donor.The production of free triiodothyronine (FT3) and free thyroxine (FT4) and the release of thyroglobulin (Tg) were measured with radioimmunoassays.Results The expression of CD40 on infiltrated lymphocytes and TFC were detected in Graves' disease but not in non-toxic goiter patient tissues.Compared with no stimulation group (23.7％ ± 7.3％),the expression of CD40 on TFC increased in IFN-γ stimulation group (86.4％ ±4.6％),IL-6 stimulation group (90.0％ ± 4.2％) and TNF-αstimulation group (87.3％ ± 4.2％).Compared with the IFN-γ + mIgG group (0.75 ± 0.06),the TFC proliferation of IFN-γ + CD40 group (1.14 ±0.14) significantly increased (P ＜0.01).The levels of FT3,FT4 and Tg secretion of IFN-γ +CD40 group were (1.10 ±0.15) pmol/L,(0.80 ±0.14) pmol/L and (30.23 ± 1.60) μg/L respectively.They were all significantly increased compared with the IFN-γ + mIgG group,of which the FT3,FT4 and Tg production were (0.76 ±0.07) pmoL/L,(0.63 ±0.09) pmol/L and (21.37 ±3.22) μg/L respectively (all P ＜ 0.05).Conclusions CD40 is abnormally expressed in thyroid tissue of Graves' disease patients.And its costimulatory signal may take part in the immunopathologic mechanism of Graves' disease.