中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
11期
974-980
,共7页
Graves病%动物模型%免疫/方法%近交系BALB/c小鼠%促甲状腺素受体/血%甲状腺素/血%重组质粒%甲状腺相关眼病
Graves病%動物模型%免疫/方法%近交繫BALB/c小鼠%促甲狀腺素受體/血%甲狀腺素/血%重組質粒%甲狀腺相關眼病
Graves병%동물모형%면역/방법%근교계BALB/c소서%촉갑상선소수체/혈%갑상선소/혈%중조질립%갑상선상관안병
Graves disease%Disease models,animal%Immunization/methods%Mice,inbred BALB/c%Receptors,thyrotropin/blood%Thyroxine/blood%Recombinant plasmid%Thyroid-associated ophthalmopathy
背景 甲状腺相关眼病(TAO)是眼科临床常见的难治性疾病,发病率在眼眶疾病中居首位.目前对于TAO的研究缺乏可复制的动物模型,在疾病早期阶段难以获得眼眶组织,具体病因及确切的发病机制仍未得到阐明.要详细了解TAO的发病机制,探寻有效防治措施,关键在于建立适当的动物模型. 目的采用脂质体包裹的促甲状腺激素受体(TSHR)胞外段基因重组质粒免疫同系雌性BALB/c小鼠,探讨该方法建立TAO动物模型的可行性. 方法 按照计算机随机分组方法将32只同系6～8周龄雌性BALB/c小鼠随机分为空白对照组、空质粒注射组、脂质体注射组和重组质粒注射组.分别于0、3、6周免疫各组小鼠,重组质粒注射组小鼠经双胫前肌和腹腔内分别注射阳离子脂质体包裹的pcDNA3.1 +/hTSHR289 30 μg和40 μg;脂质体注射组小鼠分别经双胫前肌和腹腔内分别注射未包裹重组质粒的阳离子脂质体30 μg和40 μg;空质粒注射组小鼠分别经双胫前肌和腹腔内分别注射pcDNA3.1+空质粒30 μg和40 μg;空白对照组小鼠未行任何干预.分别于初次免疫前及初次免疫后1、2、3、4个月测量各组小鼠的体质量,观察小鼠外眼表现.于初次免疫后17周末处死小鼠,取甲状腺及眼眶组织进行组织病理学观察;收集各组小鼠心脏血0.6 ～0.8 ml,采用ELISA法测定小鼠血清总甲状腺素4(TT4)和促甲状腺激素(TSH)的质量浓度. 结果 重组质粒注射组6只小鼠12只眼出现眼球突出、眼睑肿胀和角膜溃疡.空白对照组、脂质体注射组、空质粒注射组小鼠随着时间的延长体质量逐渐增加,而重组质粒注射组小鼠体质量则逐渐下降,总体比较差异有统计学意义(F时间=3.838,P=0.023),不同组间小鼠体质量总体比较差异有统计学意义(F分组=3.425,P=0.028),其中注射后2、3、4个月重组质粒注射组小鼠体质量明显低于空白对照组、空质粒注射组和脂质体注射组,差异均有统计学意义(均P＜0.05).空白对照组、脂质体注射组、空质粒注射组和重组质粒注射组小鼠血清TT4质量浓度分别为(7.75±1.00)、(7.96±0.76)、(6.76±1.10)和(4.43±2.88) μg/dl,TSH质量浓度分别为(6.36±2.58)、(4.83±3.96)、(6.63±1.71)和(1.60±1.76) ng/ml,总体比较差异均有统计学意义(F=7.150,P＜0.001;F=5.521,P＜0.01),其中重组质粒注射组小鼠血清中TT4和TSH质量浓度均明显低于空白对照组、脂质体注射组、空质粒注射组,差异均有统计学意义(均P＜0.05).组织病理学检查显示,6只重组质粒注射组小鼠甲状腺出现淋巴细胞浸润,15只眼小鼠眼眶组织出现眼眶内脂肪组织增生、淋巴细胞及肥大细胞浸润、透明质酸沉积以及眼外肌肌纤维肿胀、变性和断裂,并伴有炎性细胞浸润.结论 采用脂质体包裹的TSHR胞外段基因重组质粒免疫同系雌性BALB/c小鼠建立TAO动物模型是一种可行、有效的方法,该模型与人TAO的病理组织学特征相似,成模率高.
揹景 甲狀腺相關眼病(TAO)是眼科臨床常見的難治性疾病,髮病率在眼眶疾病中居首位.目前對于TAO的研究缺乏可複製的動物模型,在疾病早期階段難以穫得眼眶組織,具體病因及確切的髮病機製仍未得到闡明.要詳細瞭解TAO的髮病機製,探尋有效防治措施,關鍵在于建立適噹的動物模型. 目的採用脂質體包裹的促甲狀腺激素受體(TSHR)胞外段基因重組質粒免疫同繫雌性BALB/c小鼠,探討該方法建立TAO動物模型的可行性. 方法 按照計算機隨機分組方法將32隻同繫6～8週齡雌性BALB/c小鼠隨機分為空白對照組、空質粒註射組、脂質體註射組和重組質粒註射組.分彆于0、3、6週免疫各組小鼠,重組質粒註射組小鼠經雙脛前肌和腹腔內分彆註射暘離子脂質體包裹的pcDNA3.1 +/hTSHR289 30 μg和40 μg;脂質體註射組小鼠分彆經雙脛前肌和腹腔內分彆註射未包裹重組質粒的暘離子脂質體30 μg和40 μg;空質粒註射組小鼠分彆經雙脛前肌和腹腔內分彆註射pcDNA3.1+空質粒30 μg和40 μg;空白對照組小鼠未行任何榦預.分彆于初次免疫前及初次免疫後1、2、3、4箇月測量各組小鼠的體質量,觀察小鼠外眼錶現.于初次免疫後17週末處死小鼠,取甲狀腺及眼眶組織進行組織病理學觀察;收集各組小鼠心髒血0.6 ～0.8 ml,採用ELISA法測定小鼠血清總甲狀腺素4(TT4)和促甲狀腺激素(TSH)的質量濃度. 結果 重組質粒註射組6隻小鼠12隻眼齣現眼毬突齣、眼瞼腫脹和角膜潰瘍.空白對照組、脂質體註射組、空質粒註射組小鼠隨著時間的延長體質量逐漸增加,而重組質粒註射組小鼠體質量則逐漸下降,總體比較差異有統計學意義(F時間=3.838,P=0.023),不同組間小鼠體質量總體比較差異有統計學意義(F分組=3.425,P=0.028),其中註射後2、3、4箇月重組質粒註射組小鼠體質量明顯低于空白對照組、空質粒註射組和脂質體註射組,差異均有統計學意義(均P＜0.05).空白對照組、脂質體註射組、空質粒註射組和重組質粒註射組小鼠血清TT4質量濃度分彆為(7.75±1.00)、(7.96±0.76)、(6.76±1.10)和(4.43±2.88) μg/dl,TSH質量濃度分彆為(6.36±2.58)、(4.83±3.96)、(6.63±1.71)和(1.60±1.76) ng/ml,總體比較差異均有統計學意義(F=7.150,P＜0.001;F=5.521,P＜0.01),其中重組質粒註射組小鼠血清中TT4和TSH質量濃度均明顯低于空白對照組、脂質體註射組、空質粒註射組,差異均有統計學意義(均P＜0.05).組織病理學檢查顯示,6隻重組質粒註射組小鼠甲狀腺齣現淋巴細胞浸潤,15隻眼小鼠眼眶組織齣現眼眶內脂肪組織增生、淋巴細胞及肥大細胞浸潤、透明質痠沉積以及眼外肌肌纖維腫脹、變性和斷裂,併伴有炎性細胞浸潤.結論 採用脂質體包裹的TSHR胞外段基因重組質粒免疫同繫雌性BALB/c小鼠建立TAO動物模型是一種可行、有效的方法,該模型與人TAO的病理組織學特徵相似,成模率高.
배경 갑상선상관안병(TAO)시안과림상상견적난치성질병,발병솔재안광질병중거수위.목전대우TAO적연구결핍가복제적동물모형,재질병조기계단난이획득안광조직,구체병인급학절적발병궤제잉미득도천명.요상세료해TAO적발병궤제,탐심유효방치조시,관건재우건립괄당적동물모형. 목적채용지질체포과적촉갑상선격소수체(TSHR)포외단기인중조질립면역동계자성BALB/c소서,탐토해방법건립TAO동물모형적가행성. 방법 안조계산궤수궤분조방법장32지동계6～8주령자성BALB/c소서수궤분위공백대조조、공질립주사조、지질체주사조화중조질립주사조.분별우0、3、6주면역각조소서,중조질립주사조소서경쌍경전기화복강내분별주사양리자지질체포과적pcDNA3.1 +/hTSHR289 30 μg화40 μg;지질체주사조소서분별경쌍경전기화복강내분별주사미포과중조질립적양리자지질체30 μg화40 μg;공질립주사조소서분별경쌍경전기화복강내분별주사pcDNA3.1+공질립30 μg화40 μg;공백대조조소서미행임하간예.분별우초차면역전급초차면역후1、2、3、4개월측량각조소서적체질량,관찰소서외안표현.우초차면역후17주말처사소서,취갑상선급안광조직진행조직병이학관찰;수집각조소서심장혈0.6 ～0.8 ml,채용ELISA법측정소서혈청총갑상선소4(TT4)화촉갑상선격소(TSH)적질량농도. 결과 중조질립주사조6지소서12지안출현안구돌출、안검종창화각막궤양.공백대조조、지질체주사조、공질립주사조소서수착시간적연장체질량축점증가,이중조질립주사조소서체질량칙축점하강,총체비교차이유통계학의의(F시간=3.838,P=0.023),불동조간소서체질량총체비교차이유통계학의의(F분조=3.425,P=0.028),기중주사후2、3、4개월중조질립주사조소서체질량명현저우공백대조조、공질립주사조화지질체주사조,차이균유통계학의의(균P＜0.05).공백대조조、지질체주사조、공질립주사조화중조질립주사조소서혈청TT4질량농도분별위(7.75±1.00)、(7.96±0.76)、(6.76±1.10)화(4.43±2.88) μg/dl,TSH질량농도분별위(6.36±2.58)、(4.83±3.96)、(6.63±1.71)화(1.60±1.76) ng/ml,총체비교차이균유통계학의의(F=7.150,P＜0.001;F=5.521,P＜0.01),기중중조질립주사조소서혈청중TT4화TSH질량농도균명현저우공백대조조、지질체주사조、공질립주사조,차이균유통계학의의(균P＜0.05).조직병이학검사현시,6지중조질립주사조소서갑상선출현림파세포침윤,15지안소서안광조직출현안광내지방조직증생、림파세포급비대세포침윤、투명질산침적이급안외기기섬유종창、변성화단렬,병반유염성세포침윤.결론 채용지질체포과적TSHR포외단기인중조질립면역동계자성BALB/c소서건립TAO동물모형시일충가행、유효적방법,해모형여인TAO적병리조직학특정상사,성모솔고.
Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease,and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed.Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models.Methods Thirty-two 6-to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1 +/hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3,6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month,2,3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection,and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice,and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH)were assayed by ELISA.Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup =3.425, P =0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group,liposomes injection group (Ftime =0.838 ,P=0.023).The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88) μg/dlin the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58),(4.83±3.96),(6.63±1.71) and (1.60 ±1.76) ng/ml, showing significant differences among the groups (F =7.150, P＜0.001;F =5.521, P＜0.01) , and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group,liposomes injection group and blank plasmid group (all at P ＜ 0.05).Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes,deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group.Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1 +/hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO.
