中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
7期
593-598
,共6页
陈杰%孙卫民%宋斗%张超雄%王金泉%徐晓%陶君
陳傑%孫衛民%宋鬥%張超雄%王金泉%徐曉%陶君
진걸%손위민%송두%장초웅%왕금천%서효%도군
葡萄膜视网膜炎/免疫%获得性免疫%自身免疫%内毒素%百日咳毒素%小鼠,C57BL/6(H-2b)
葡萄膜視網膜炎/免疫%穫得性免疫%自身免疫%內毒素%百日咳毒素%小鼠,C57BL/6(H-2b)
포도막시망막염/면역%획득성면역%자신면역%내독소%백일해독소%소서,C57BL/6(H-2b)
Uveoretinitis/immunology%Adaptive immunity%Autoimmunity%Endotoxin%Pertussis toxin%Mouse,C57BL/6(H-2b)
背景 研究表明,使用百日咳毒素(PTX)诱导的传统实验性自身免疫性葡萄膜视网膜炎(EAU)模型在病原学和流行病学上与人类葡萄膜炎的发病环境有明显差异,且PTX本身可影响免疫应答.我们先前的研究已成功建立了内毒素——脂多糖(LPS)诱导的EAU模型,但PTX与LPS在EAU模型中的不同作用尚不清楚. 目的 比较在不同免疫阶段注射PTX和LPS对EAU诱导的不同影响,探讨LPS和PTX在葡萄膜视网膜炎中的作用机制. 方法 将SPF级6～8周龄C57BL/6(H-2 b)小鼠20只采用随机数字表法随机分为0 d-PTX-EAU组、7 d-PTX-EAU组、0 d-LPS-EAU组和7 d-LPS-EAU组,分别于人类光感受器间维生素A类结合蛋白多肽片段1-20(IRBP 1-20)及完全弗氏佐剂(CFA)免疫小鼠后即刻或第7天在小鼠足底注射10 g/L LPS 30 μl或腹腔内注射1.0 mg/L PTX 0.5 ml,分别建立PTX和LPS诱导的EAU模型.于免疫后第19天,耳廓皮内分别注射IRBP 1-20,48 h测量模型鼠耳廓厚度,比较两种模型小鼠的迟发型超敏反应(DTH);制备小鼠脾脏细胞匀浆,用3H脱氧胸苷(3H TdR)掺入法比较各组小鼠的特异性淋巴细胞增生反应;收集小鼠视网膜组织行苏木精-伊红染色,观察各组小鼠视网膜的炎症表现并参照Caspi标准进行炎症评分,对不同模型组小鼠的检测结果进行比较. 结果 0 d-PTX-EAU组和7 d-LPS-EAU组小鼠均出现典型的葡萄膜视网膜炎病理损害,可见明显的炎性细胞浸润、视网膜皱褶和脱离及视网膜全层结构排列紊乱,0 d-PTX-EAU组小鼠玻璃体炎和肉芽肿反应略重于7 d-LPS-EAU组;而7 d-PTX-EAU组小鼠仅见视网膜血管轻度扩张,0 d-LPS-EAU组小鼠可见少量视网膜血管扩张和个别区域的视网膜折叠.0 d-PTX-EAU组和7 d-LPS-EAU组小鼠的EAU评分明显高于7 d-PTX-EAU组和0 d-LPS-EAU组,差异均有统计学意义(P＜0.05).0 d-PTX-EAU组和7 d-PTX-EAU组小鼠耳廓厚度分别为(62.600±3.362) μm和(60.000±2.345) μm,均明显高于0 d-LPS-EAU组的(30.400±1.817) μm和7 d-LPS-EAU组小鼠的(32.800±1.643) μm,4个组间的总体差异有统计学意义(F分组=259.751,P=0.000).0 d-PTX-EAU组和7 d-PTX-EAU组小鼠体外培养的淋巴细胞克隆数显著增加,其3H TdR掺入值(cpm)分别为(16 150.000±799.218)/min和(16 120.000±729.383)/min,均明显高于0d-LPS-EAU组的(8 348.000±258.979)/min和7 d-LPS-EAU组的(8 540.000±81.548)/min,4个组间小鼠淋巴细胞增生的差异有统计学意义(F分组=316.978,P=0.000). 结论 免疫的同时注射PTX和免疫后第7天注射LPS均可诱导典型的EAU,LPS在EAU中的作用与PTX不同,主要作用于免疫的效应阶段,可能与血-眼屏障的破坏有关.
揹景 研究錶明,使用百日咳毒素(PTX)誘導的傳統實驗性自身免疫性葡萄膜視網膜炎(EAU)模型在病原學和流行病學上與人類葡萄膜炎的髮病環境有明顯差異,且PTX本身可影響免疫應答.我們先前的研究已成功建立瞭內毒素——脂多糖(LPS)誘導的EAU模型,但PTX與LPS在EAU模型中的不同作用尚不清楚. 目的 比較在不同免疫階段註射PTX和LPS對EAU誘導的不同影響,探討LPS和PTX在葡萄膜視網膜炎中的作用機製. 方法 將SPF級6～8週齡C57BL/6(H-2 b)小鼠20隻採用隨機數字錶法隨機分為0 d-PTX-EAU組、7 d-PTX-EAU組、0 d-LPS-EAU組和7 d-LPS-EAU組,分彆于人類光感受器間維生素A類結閤蛋白多肽片段1-20(IRBP 1-20)及完全弗氏佐劑(CFA)免疫小鼠後即刻或第7天在小鼠足底註射10 g/L LPS 30 μl或腹腔內註射1.0 mg/L PTX 0.5 ml,分彆建立PTX和LPS誘導的EAU模型.于免疫後第19天,耳廓皮內分彆註射IRBP 1-20,48 h測量模型鼠耳廓厚度,比較兩種模型小鼠的遲髮型超敏反應(DTH);製備小鼠脾髒細胞勻漿,用3H脫氧胸苷(3H TdR)摻入法比較各組小鼠的特異性淋巴細胞增生反應;收集小鼠視網膜組織行囌木精-伊紅染色,觀察各組小鼠視網膜的炎癥錶現併參照Caspi標準進行炎癥評分,對不同模型組小鼠的檢測結果進行比較. 結果 0 d-PTX-EAU組和7 d-LPS-EAU組小鼠均齣現典型的葡萄膜視網膜炎病理損害,可見明顯的炎性細胞浸潤、視網膜皺褶和脫離及視網膜全層結構排列紊亂,0 d-PTX-EAU組小鼠玻璃體炎和肉芽腫反應略重于7 d-LPS-EAU組;而7 d-PTX-EAU組小鼠僅見視網膜血管輕度擴張,0 d-LPS-EAU組小鼠可見少量視網膜血管擴張和箇彆區域的視網膜摺疊.0 d-PTX-EAU組和7 d-LPS-EAU組小鼠的EAU評分明顯高于7 d-PTX-EAU組和0 d-LPS-EAU組,差異均有統計學意義(P＜0.05).0 d-PTX-EAU組和7 d-PTX-EAU組小鼠耳廓厚度分彆為(62.600±3.362) μm和(60.000±2.345) μm,均明顯高于0 d-LPS-EAU組的(30.400±1.817) μm和7 d-LPS-EAU組小鼠的(32.800±1.643) μm,4箇組間的總體差異有統計學意義(F分組=259.751,P=0.000).0 d-PTX-EAU組和7 d-PTX-EAU組小鼠體外培養的淋巴細胞剋隆數顯著增加,其3H TdR摻入值(cpm)分彆為(16 150.000±799.218)/min和(16 120.000±729.383)/min,均明顯高于0d-LPS-EAU組的(8 348.000±258.979)/min和7 d-LPS-EAU組的(8 540.000±81.548)/min,4箇組間小鼠淋巴細胞增生的差異有統計學意義(F分組=316.978,P=0.000). 結論 免疫的同時註射PTX和免疫後第7天註射LPS均可誘導典型的EAU,LPS在EAU中的作用與PTX不同,主要作用于免疫的效應階段,可能與血-眼屏障的破壞有關.
배경 연구표명,사용백일해독소(PTX)유도적전통실험성자신면역성포도막시망막염(EAU)모형재병원학화류행병학상여인류포도막염적발병배경유명현차이,차PTX본신가영향면역응답.아문선전적연구이성공건립료내독소——지다당(LPS)유도적EAU모형,단PTX여LPS재EAU모형중적불동작용상불청초. 목적 비교재불동면역계단주사PTX화LPS대EAU유도적불동영향,탐토LPS화PTX재포도막시망막염중적작용궤제. 방법 장SPF급6～8주령C57BL/6(H-2 b)소서20지채용수궤수자표법수궤분위0 d-PTX-EAU조、7 d-PTX-EAU조、0 d-LPS-EAU조화7 d-LPS-EAU조,분별우인류광감수기간유생소A류결합단백다태편단1-20(IRBP 1-20)급완전불씨좌제(CFA)면역소서후즉각혹제7천재소서족저주사10 g/L LPS 30 μl혹복강내주사1.0 mg/L PTX 0.5 ml,분별건립PTX화LPS유도적EAU모형.우면역후제19천,이곽피내분별주사IRBP 1-20,48 h측량모형서이곽후도,비교량충모형소서적지발형초민반응(DTH);제비소서비장세포균장,용3H탈양흉감(3H TdR)참입법비교각조소서적특이성림파세포증생반응;수집소서시망막조직행소목정-이홍염색,관찰각조소서시망막적염증표현병삼조Caspi표준진행염증평분,대불동모형조소서적검측결과진행비교. 결과 0 d-PTX-EAU조화7 d-LPS-EAU조소서균출현전형적포도막시망막염병리손해,가견명현적염성세포침윤、시망막추습화탈리급시망막전층결구배렬문란,0 d-PTX-EAU조소서파리체염화육아종반응략중우7 d-LPS-EAU조;이7 d-PTX-EAU조소서부견시망막혈관경도확장,0 d-LPS-EAU조소서가견소량시망막혈관확장화개별구역적시망막절첩.0 d-PTX-EAU조화7 d-LPS-EAU조소서적EAU평분명현고우7 d-PTX-EAU조화0 d-LPS-EAU조,차이균유통계학의의(P＜0.05).0 d-PTX-EAU조화7 d-PTX-EAU조소서이곽후도분별위(62.600±3.362) μm화(60.000±2.345) μm,균명현고우0 d-LPS-EAU조적(30.400±1.817) μm화7 d-LPS-EAU조소서적(32.800±1.643) μm,4개조간적총체차이유통계학의의(F분조=259.751,P=0.000).0 d-PTX-EAU조화7 d-PTX-EAU조소서체외배양적림파세포극륭수현저증가,기3H TdR참입치(cpm)분별위(16 150.000±799.218)/min화(16 120.000±729.383)/min,균명현고우0d-LPS-EAU조적(8 348.000±258.979)/min화7 d-LPS-EAU조적(8 540.000±81.548)/min,4개조간소서림파세포증생적차이유통계학의의(F분조=316.978,P=0.000). 결론 면역적동시주사PTX화면역후제7천주사LPS균가유도전형적EAU,LPS재EAU중적작용여PTX불동,주요작용우면역적효응계단,가능여혈-안병장적파배유관.
Background Researches indicated that etiology and epidemiology of pertussis toxin (PTX)dependent experimental autoimmune uveoretinitis(EAU)model are very different with human uveoretinitis owing to the influence of PTX on immune.Our previous study has established lipopolysaccharide (LPS),an endotoxin,which instesad of PTX,mediated EAU model.However,the exact roles of LPS and PTX in EAU still remained unclear.Objective This study was to investigate the roles of LPS and PTX in EAU model.Methods Twenty SPF C57BL/6(H-2b) mice were assigned to 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group using random number table method.The mice were immunized with interphotoreceptor retinoid-binding protein 1-20(IRBP 1-20) emulsified in complete Freund adjuvant (CFA),and concurrently with or on day 7 postimmunization,LPS or PTX was injected in the footpad or intraperitoneally respectively.Delayed-type hypersensitivity (DTH) of the mice was evaluated by measuring the ear thickness 48 hours after IRBP was injected into the ear pinna,and lymphocyte proliferation was assessed by tritiated thymidine uptake.Retinal histopathological examination was performed and scored based on criteria of Caspi.The use and care of experimental animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Serious infiltration of inflammatory cells,disorder of entire retinal structure and retinal folds were seen in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group on 21 days after injection of PTX or 14 days after injection of LPS,and severe vitritis and a few granuloma-like lesions were found in the 0 d-PTX-EAU group.However,only mild vasodilatation or less retinal folds were found in the 7 d-PTX-EAU group and 0 d-LPS-EAU group.The pathological scores in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group were higher than those of the 7 d-PTX-EAU group and 0 d-LPS-EAU group (all at P ＜ 0.05).The ear thickness was (62.600 ± 3.362) μm,(60.000±2.345) μm,(30.400± 1.817) μm and (32.800 ± 1.643) μm in the 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group,showing a significantly difference among the 4 groups (Fgroup =259.751,P=0.000),and the ear thicknesses of 0 d-PTX-EAU group and 7 d-PTX-EAU group were significantly higher than those of the 0 d-LPS-EAU group and 7 d-LPS-EAU group (all at P＜0.05).The lymphocyte proliferation was strongly enhanced in PTX-EAU groups,and the radiation count per minute (cpm) was (16 150.000±799.218)/min and (16 120.000±729.383)/min in the 0 d-PTX EAU group and 7 d-PTX EAU group,and (8 348.000±258.979)/min and (8 540.000±81.548)/min in the 0 d-LPS EAU group and 7 d-LPS EAU group respectively,with a significant difference among the PTX-EAU groups and LPS-EAU groups (Fgroup =316.978,P=0.000).Conclusions LPS and PTX play different roles during the EAU formation.LPS may be involved in the breakdown of blood-retina barriers (BRB).
