摘要
自身免疫性甲状腺疾病(autoimmune thyroid disease,AITD)是一组发病机制复杂的器官特异性自身免疫疾病。人促甲状腺激素受体(hTSHR)是AITD的主要靶抗原,在AITD的发病中起重要作用。hTSHR基因定位于14号染色体(14q31)上,是一种G蛋白偶联受体,为大分子糖蛋白其肽链可分为膜外区,跨膜区和膜内区。TSHR膜外区(TSHR-ecd)为促甲状腺激素(TSH)或TSHR抗体(TRAb)识别及结合部位,有广泛的、多个不连续的结合位点。其中TSHR刺激型抗体(TSAb)的抗原决定簇存在于膜外区的N端(氨基酸3437,40,4245,5256),TSHR刺激阻断型抗体(TSBAb)的抗原决定簇存在于膜外区的C端(氨基酸301,385,390),而这些区域也都是TSH的结合位点。TSH主要作用的靶器官是甲状腺,它是通过与甲状腺细胞膜上的TSH受体TSHR结合,通过CAMP第二信号系统引起一系列生物学反应,促进甲状腺激素的分泌,提高甲状腺的摄碘能力并促进甲状腺激素的合成、释放,对甲状腺组织的生长、分化等起着非常重要的作用。TSHR属甲状腺的正常组分,但是,遗传因素的存在和免疫因素、环境因素的诱发,引起了hTSHR的变异,使其产生了抗原性,致TSHR特异性免疫原肽得以暴露,破坏了人体对TSHR的耐受性,产生大量的TRAb,引发免疫应答而致病。TRAb的检测在AITD的科研及临床中有着极其重要的意义。
目的
以本室制备富含TSAb抗原决定簇的重组TrxTSHRn蛋白、富含TSBAb抗原决定簇的重组TrxTSHRc蛋白分别作为抗原,酶联免疫吸附(ELISA)检测人血清TRAb(包括TSAb、TSBAb)含量,探讨此TRAb检测在AITD诊断和治疗中的意义。
方法
取本室研制的重组TrxTSHRn、TrxTSHRc蛋白为抗原,通过不同的抗原包被量、不同的血清稀释度优化,建立TSAb、TSBAb的间接ELISA检测方法。经精密度测试后,用于检测不同年龄、性别组健康人群,及各甲状腺疾病患者血清中的TRAb。
结果
1.以TrxTSHRc蛋白为抗原TRAb检测,抗原包被量16ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.325和0.358,批内变异cv%为3.77%和3.42%,批间变异cv%为4.87%和5.12%,正常人中OD405值为0.319±0.107,正常值范围为0.105~0.533,正常人组阳性率3.30%(9/272);Hashimoto甲状腺炎患者阳性率66.01%(336/509),其中伴甲状腺功能减退者阳性率77.78%(252/324)、伴甲状腺功能亢进者52.94%(63/119)、甲功正常者31.82%(21/66);初发Graves'患者阳性率41.86%(44/105);治疗1年Graves'患者阳性率17.86%(5/28);单纯性甲状腺肿患者阳性率6.98%(3/43);亚急性甲状腺炎患者阳性率11.1%(5/45);非毒性结节性甲状腺肿患者阳性率8.70%(6/69);甲状腺腺瘤患者阳性率3.2%(1/31)。
2.以TrxTSHRn蛋白为抗原TRAb检测,抗原包被量100ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.098和0.410,批内变异为3.53%和4.12%,批间变异为4.52%和4.67%,正常人群中儿童组共115例(0至14岁,平均3.8岁)OD405为0.186±0.094,正常成人组1060例(18至81岁)OD405值为0.398±0.167,二者差异有统计学意义,性别组之间及成人组各年龄段之间差异无统计学意义。成人组正常值范围0.064~0.732,阳性率2.45%(26/1060)、初发Graves'患者阳性率70.31%(74/105)、治疗1年Graves'患者阳性率35.7%(10/28)、Hashimoto甲状腺炎患者阳性率36.67%(44/120)、单纯性甲状腺肿患者阳性率4.6%(2/43)、亚急性甲状腺炎患者阳性率6.67%(3/45)、非毒性结节性甲状腺肿患者阳性率7.25%(5/69)。
结论
1.以TrxTSHRc蛋白为抗原ELISA法检测TRAb对AITD,尤其是Hashimoto甲状腺炎伴甲减的诊断中有重要意义。
2.以TrxTSHRn蛋白为抗原ELISA法检测TRAb值范围在儿童组,成人年组存在差异,有统计学意义,在男、女性别组差异无统计学意义。
3.以TrxTSHRn蛋白为抗原ELISA法检测TRAb对AITD,尤其是Graves'病的诊断中有重要意义。
Autoimmune thyroid disease(AITD)is a sort of organ specificity autoimmune disease.Human thyroid stimulating hormone receptor is a primary target antigen in AITD.And plays a role in set and process of AITD.The hTSHR gene assignment at No.14 chromatin body(14q31),is a G protein-coupled large moleculer glycoprotein consisting of extracellular(TSHR-ecd),transmembrane and intramembrane domain.TSHR-ecd has multi-discontinuous binding site where the thyrotropic-stimulating hormone(TSH)and thyrotrophin receptor antibody(TRAb) can discriminate and combine.Epitopes of thyroid-stimulating antibody(TSAb) mainly lie in N terminal of TSHR-ecd(amino acid3437,40,4245,5256),while thyroid stimulating blocking antibody(TSBAb)in C terminal(amino acid 301,385,390).TSH's target organ is thyroid gland.TSH can combin with TSHR and cause a series of biochemical event by CAMP second signal system.Physiologically, the combination can regulate the normal function and growth of thyroid follicular epithelial cell.TSHR is the normal composition of the thyroid gland.But under the function of heredity and environment,the structure of TSHR changes and has the antigenicity which can cause autoimmune thyroid disease(AITD)and product autoantibodies(TRAb).The detection of TRAb is very important for the dignosis, treatment and prognostic evaluation.
Objective:
Using Recombinent TrxTSHRn Protein which contains TSAb antigen determinant.and TrxTSHRc Protein which contains TSBAb antigen determinant as antigen,establish enzyme linked immunosorbent assay(ELISA).for detecting the TRAb in the serum of health and thyroid disease patient.Investigate this detection's significance for the dignosis,treatment in AITD。
Method:
Using Recombinent TrxTSHRn Protein and TrxTSHRc Protein as antigen, determin the optimum condition,establish the indirect ELISA for detecting the TRAb in the serum of health and thyroid disease patient.
Result:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 16ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.5%,4.12%respectively.The interassay CV values are 4.52%,4.67%.The mean±standard deviation(SD)of normal value is 0.319±0.107(in adult).The positive rate of untreanted Graves' disease are 41.86%,treated Graves' disease are 7.86%,Hashimoto thyroiditis 66.01%(hypothyroidism77.78% hyperthyroidism52.94%),simple goiter are 6.98%,subacute thyroiditis are 11.1%Non-toxic nodular goiter;nontoxic nodular goiter are 8.7%thyroid adenoma are 3.1%respectively.
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 100ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.77%,3.42%respectively.The interassay CV values are 4.87%,5.12%.The mean±standard deviation(SD)of normal value is 0.186±0.094(in children),0.398±0.167(in adult).The positive rate of untreanted Graves' disease are 70.31%,treated Graves' disease are 35.7%,Hashimoto thyroiditis 36.67%,simple goiter are 4.6%,subacute thyroiditis are 6.67%Non-toxic nodular goiter;nontoxic nodular goiter are 7.25%respectively.
Conclusion:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen is very important for the dignosis of AITD,especially in Hashimoto thyroiditis complicate hypothyroidism;
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen in normal:There is difference in range of normal values between children and adult,but no statistically significant between male and female;
3.The detection of TRAb using recombinent TrxTSHRn protein as antigen is very important for the dignosis of AITD,especially in Graves'disease.
引文
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