目的 观察碘过量(high iodine,HI)和多聚肌苷酸-聚胞苷酸[Polyinosinic-Polycytidylic acid,Poly (I:C),Poly]及甲状腺球蛋白(Thyroglobulin,TG)诱发小鼠甲状腺炎对Toll样受体3(Toll-like receptor 3,TLR3)表达的影响,探讨TLR3在自身免疫性甲状腺炎发病中的作用.方法 NOD(Non-obese diabetic)小鼠42只,体质量(20±3)g.按体质量将小鼠随机分为6组:对照组、HI组、Poly组、TG组、HI+TG组、HI+Poly组,每组7只.对照组:饮用去离子水,腹腔注射生理盐水0.1 ml,每天1次,连续1周,在处死小鼠前1周隔日1次,同样剂量生理盐水再注射3次;HI组:饮用0.05%的碘化钠去离子水,腹腔注射生理盐水(同对照组);Poly 组:饮用去离子水,腹腔注射0.1 ml Poly(1 g/L,按5 mg/kg体质量),每天1次,连续1周,处死前1周隔日1次,同剂量Poly再注射3次;TG组:饮去离子水,腹腔注射生理盐水(同对照组),皮下免疫猪TG 0.1 mg,在喂养第4、8周时分别再加强免疫1次,剂量减半;HI+Poly组:给药方法同HI组和Poly组;HI+TG组:给药方法同HI组和TG组.喂养8周后处死小鼠,取出甲状腺组织,冰冻切片、常规HE染色,光镜下观察小鼠甲状腺组织形态学变化:根据甲状腺组织炎细胞浸润数量及浸润范围、滤泡破坏范围等进行炎症程度分级;应用TLR3抗体对甲状腺切片进行免疫荧光染色,荧光显微镜下观察TLR3的表达,体视学分析甲状腺TLR3阳性细胞数密度变化.结果光镜下,Poly组甲状腺未见炎细胞浸润,HI组和TG组小鼠甲状腺都有不同程度的炎细胞浸润,HI+TG组和HI+Poly组甲状腺炎症细胞浸润和甲状腺滤泡破坏严重,炎症分级均在"++"以上.免疫荧光显示.HI组和Poly组的甲状腺滤泡上皮细胞可见到TLR3表达,在HI组和HI+Poly组炎症区域出现TLR3表达强阳性的炎症细胞.体视学分析甲状腺TLR3阳性细胞数密度,对照组、HI组、Poly组、TG组、HI+TG组、HI+Poly组组间比较差异有统计学意义(F=7.870,P<0.01);与对照组[(0.062±0.025)mm2]比较,HI+Poly组[(9.287±0.522)mm2]增加最为显著(P<0.01),而且HI+Poly组高于HI组[(2.570±0.257)mm2]和Poly组[(1.361±0.148)mm2,P均<0.01],HI+TG组[(4.843±0.405)mm2]高于HI组和TG组[(1.601±0.268)mm2,P均<0.01].结论 HI和TG免疫可诱发NOD鼠发生甲状腺炎,并刺激甲状腺滤泡上皮表达TLR3,Poly加重了HI诱发的NOD鼠甲状腺炎的病理变化过程;浸润的炎症细胞中亦有TLR3强阳性的细胞,提示TLR3途径参与了自身免疫性甲状腺炎的发病过程.
目的 觀察碘過量(high iodine,HI)和多聚肌苷痠-聚胞苷痠[Polyinosinic-Polycytidylic acid,Poly (I:C),Poly]及甲狀腺毬蛋白(Thyroglobulin,TG)誘髮小鼠甲狀腺炎對Toll樣受體3(Toll-like receptor 3,TLR3)錶達的影響,探討TLR3在自身免疫性甲狀腺炎髮病中的作用.方法 NOD(Non-obese diabetic)小鼠42隻,體質量(20±3)g.按體質量將小鼠隨機分為6組:對照組、HI組、Poly組、TG組、HI+TG組、HI+Poly組,每組7隻.對照組:飲用去離子水,腹腔註射生理鹽水0.1 ml,每天1次,連續1週,在處死小鼠前1週隔日1次,同樣劑量生理鹽水再註射3次;HI組:飲用0.05%的碘化鈉去離子水,腹腔註射生理鹽水(同對照組);Poly 組:飲用去離子水,腹腔註射0.1 ml Poly(1 g/L,按5 mg/kg體質量),每天1次,連續1週,處死前1週隔日1次,同劑量Poly再註射3次;TG組:飲去離子水,腹腔註射生理鹽水(同對照組),皮下免疫豬TG 0.1 mg,在餵養第4、8週時分彆再加彊免疫1次,劑量減半;HI+Poly組:給藥方法同HI組和Poly組;HI+TG組:給藥方法同HI組和TG組.餵養8週後處死小鼠,取齣甲狀腺組織,冰凍切片、常規HE染色,光鏡下觀察小鼠甲狀腺組織形態學變化:根據甲狀腺組織炎細胞浸潤數量及浸潤範圍、濾泡破壞範圍等進行炎癥程度分級;應用TLR3抗體對甲狀腺切片進行免疫熒光染色,熒光顯微鏡下觀察TLR3的錶達,體視學分析甲狀腺TLR3暘性細胞數密度變化.結果光鏡下,Poly組甲狀腺未見炎細胞浸潤,HI組和TG組小鼠甲狀腺都有不同程度的炎細胞浸潤,HI+TG組和HI+Poly組甲狀腺炎癥細胞浸潤和甲狀腺濾泡破壞嚴重,炎癥分級均在"++"以上.免疫熒光顯示.HI組和Poly組的甲狀腺濾泡上皮細胞可見到TLR3錶達,在HI組和HI+Poly組炎癥區域齣現TLR3錶達彊暘性的炎癥細胞.體視學分析甲狀腺TLR3暘性細胞數密度,對照組、HI組、Poly組、TG組、HI+TG組、HI+Poly組組間比較差異有統計學意義(F=7.870,P<0.01);與對照組[(0.062±0.025)mm2]比較,HI+Poly組[(9.287±0.522)mm2]增加最為顯著(P<0.01),而且HI+Poly組高于HI組[(2.570±0.257)mm2]和Poly組[(1.361±0.148)mm2,P均<0.01],HI+TG組[(4.843±0.405)mm2]高于HI組和TG組[(1.601±0.268)mm2,P均<0.01].結論 HI和TG免疫可誘髮NOD鼠髮生甲狀腺炎,併刺激甲狀腺濾泡上皮錶達TLR3,Poly加重瞭HI誘髮的NOD鼠甲狀腺炎的病理變化過程;浸潤的炎癥細胞中亦有TLR3彊暘性的細胞,提示TLR3途徑參與瞭自身免疫性甲狀腺炎的髮病過程.
목적 관찰전과량(high iodine,HI)화다취기감산-취포감산[Polyinosinic-Polycytidylic acid,Poly (I:C),Poly]급갑상선구단백(Thyroglobulin,TG)유발소서갑상선염대Toll양수체3(Toll-like receptor 3,TLR3)표체적영향,탐토TLR3재자신면역성갑상선염발병중적작용.방법 NOD(Non-obese diabetic)소서42지,체질량(20±3)g.안체질량장소서수궤분위6조:대조조、HI조、Poly조、TG조、HI+TG조、HI+Poly조,매조7지.대조조:음용거리자수,복강주사생리염수0.1 ml,매천1차,련속1주,재처사소서전1주격일1차,동양제량생리염수재주사3차;HI조:음용0.05%적전화납거리자수,복강주사생리염수(동대조조);Poly 조:음용거리자수,복강주사0.1 ml Poly(1 g/L,안5 mg/kg체질량),매천1차,련속1주,처사전1주격일1차,동제량Poly재주사3차;TG조:음거리자수,복강주사생리염수(동대조조),피하면역저TG 0.1 mg,재위양제4、8주시분별재가강면역1차,제량감반;HI+Poly조:급약방법동HI조화Poly조;HI+TG조:급약방법동HI조화TG조.위양8주후처사소서,취출갑상선조직,빙동절편、상규HE염색,광경하관찰소서갑상선조직형태학변화:근거갑상선조직염세포침윤수량급침윤범위、려포파배범위등진행염증정도분급;응용TLR3항체대갑상선절편진행면역형광염색,형광현미경하관찰TLR3적표체,체시학분석갑상선TLR3양성세포수밀도변화.결과광경하,Poly조갑상선미견염세포침윤,HI조화TG조소서갑상선도유불동정도적염세포침윤,HI+TG조화HI+Poly조갑상선염증세포침윤화갑상선려포파배엄중,염증분급균재"++"이상.면역형광현시.HI조화Poly조적갑상선려포상피세포가견도TLR3표체,재HI조화HI+Poly조염증구역출현TLR3표체강양성적염증세포.체시학분석갑상선TLR3양성세포수밀도,대조조、HI조、Poly조、TG조、HI+TG조、HI+Poly조조간비교차이유통계학의의(F=7.870,P<0.01);여대조조[(0.062±0.025)mm2]비교,HI+Poly조[(9.287±0.522)mm2]증가최위현저(P<0.01),이차HI+Poly조고우HI조[(2.570±0.257)mm2]화Poly조[(1.361±0.148)mm2,P균<0.01],HI+TG조[(4.843±0.405)mm2]고우HI조화TG조[(1.601±0.268)mm2,P균<0.01].결론 HI화TG면역가유발NOD서발생갑상선염,병자격갑상선려포상피표체TLR3,Poly가중료HI유발적NOD서갑상선염적병리변화과정;침윤적염증세포중역유TLR3강양성적세포,제시TLR3도경삼여료자신면역성갑상선염적발병과정.
Objective To observe the effect of iodine excess(HI),polyinosinic-polycytidylic acid[Poly(I:C),Poly]and thyroglobulin(TG)on the thyroid of mice by the expression of Toll-like receptor 3(TLR3)to reveal the functional role of TLR3 in autoimmune thyroiditis.Methods Forty-two non-obese diabetic mice,body weight (20±3)g,were divided into six groups:control group,HI group,Poly group,TG group,HI+TG group,HI+Poly group. Fed with deionized water and injected intraperitoneally with physiological saline 0.1 ml each day for a week, the mice in control group were injected with physiological saline every other day at the same dose for 1 week before they were sacrificed; HI group drank 0.05% NaI water and were injected intraperitoneally with physiological saline same as control group; Poly group drank deionized water and were injected intraperitoneally with poly 0.1 ml (1 g/L)each day of the week, then the mice were injected with Poly every other day at the same dose for 1 week before they were sacrificed; TG group drank deionized water and were injected intraperitoneally with physiological saline same as control group, immunized with 0.1 mg TG by subcutaneously injecting and the immunization was enhanced after they were fed half dose for 4 and 8 weeks separately. In HI + Poly group, the treatment was the same as HI group and Poly group; HI + TG group: the treatment was the same as HI group and TG group. Eight weeks later, mice were sacrificed and thyroids were taken to make frozen sections, Hematoxylin-Eosin (HE) staining was employed to observe the morphological change of the thyroids. The expression of TLR3 of thyroids was observed under fluorescence microscope after Immumofluorescence using TLR3 antibody and TR3-positive cells were analyzed in the thyroid density. Results HE staining showed thyroids of Poly group had no inflammation under microscope.There were different degrees of inflammatory cell infiltration in HI group and TG group. The inflammatory cell infiltration and the damage of follicular thyroid of HI + TG group and HI + Poly group were serious, and the degrees of inflammation were higher over "++". Thyroid follicular epithelial cell with TLR3 expression could be seen in Poly group and HI group, meanwhile, there were TLR3 strong positive inflammatory cells in HI group under fluorescent microscope. Using stereological analysis of TLR3-positive cell density in the thyroid, the difference between groups was statistically significant(F=7.870, P<0.01 ). TLR3-positive cell density in the thyroid of HI + Poly group was higher[ (9.287 ± 0.522)mm2] than control group[ (0.062 ± 0.025)mm2, P < 0.01] significantly, meanwhile, the density in HI + Poly group was higher than HI group [ (2.574 ± 0.257 )mm2] and Poly group[ (1.361 ± 0.148 )mm2, all P < 0.01]. The density in HI + TG group[ (4.843±0.405)mm2] was higher than HI group and TG group[(1.601 ±0.268)mm2, all P < 0.01 )]. Conclusions Excessive iodine and thyroglobulin can induce thyroiditis, and stimulate the expression of TLR3 in the thyroid follicular epithelial, Poly aggravated thyroiditis induced by iodine excess in NOD mice; TLR3 positive inflammatory cells also appeared in inflammatory region, suggesting that TLR3 is involved in the pathogenesis of autoimmune thyroiditis
