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
600-606
,共7页
徐如霞%李静%葛向红%石红霞%孟宪娴%李海燕%杨鹏霞%贾亚丁
徐如霞%李靜%葛嚮紅%石紅霞%孟憲嫻%李海燕%楊鵬霞%賈亞丁
서여하%리정%갈향홍%석홍하%맹헌한%리해연%양붕하%가아정
自身免疫性疾病%动物模型%化学诱导葡萄膜炎%化学诱导视网膜炎%骨髓间充质干细胞/治疗%曲安奈德/治疗%肿瘤坏死因子-α%玻璃体腔内注射
自身免疫性疾病%動物模型%化學誘導葡萄膜炎%化學誘導視網膜炎%骨髓間充質榦細胞/治療%麯安奈德/治療%腫瘤壞死因子-α%玻璃體腔內註射
자신면역성질병%동물모형%화학유도포도막염%화학유도시망막염%골수간충질간세포/치료%곡안내덕/치료%종류배사인자-α%파리체강내주사
Autoimmune disease%Disease model,animal%Uveitis/chemically induced%Retinitis/chemically induced%Bone marrow mesenchymal stem cell/therapy%Triamcinolone acetonide/therapy%Tumor necrosis factor-α%Intravitreal injection
背景 葡萄膜炎多由自身免疫反应所致,以往多应用糖皮质激素和免疫抑制剂进行治疗,但存在药物依赖和眼压升高的风险及其他不良反应.骨髓间充质干细胞(BMSCs)在抗炎、抑制新生血管方面具有较好的作用,且具有低免疫原性和免疫调节功能,但其在葡萄膜炎治疗方面的研究较少. 目的 比较BMSCs和曲安奈德(TA)对兔实验性自身免疫性葡萄膜炎(EAU)的治疗效果.方法 从5只新西兰大白兔股骨和胫骨骨髓中分离、培养BMSCs并进行传代,用流式细胞技术检测细胞表面抗原CD105、CD34和CD45的表达以鉴定细胞,取第3～4代细胞用于实验.将32只新西兰大白兔采用随机数字表法随机分为正常对照组、模型对照组、BMSCs干预组和TA干预组.正常对照组不进行任何处理,其余3个组用质量分数2％牛血清蛋白(BSA)静脉注射和玻璃体腔内注射法建立EAU模型.造模成功后24 h,模型对照组经右眼玻璃体腔内注射0.1 ml PBS; BMSCs干预组以同样方法注射0.1 ml BMSCs悬液,细胞密度为2×106个/ml;TA干预组注射0.1 ml(4 mg)TA.于眼部注射后15d每隔3天裂隙灯显微镜下观察实验兔的眼前节表现,并参照Caspi的标准进行炎症评分;利用眼底彩色照相法及眼部B型超声检查法观察各组兔眼后节表现;于眼部注射后30 d处死实验兔,制备眼组织标本,进行常规组织病理学检查,参照Caspi的标准进行视网膜病理学评分;于眼部注射后每隔3天取各组兔的房水0.1ml及外周血3 ml,采用ELISA法检测兔血清及房水中肿瘤坏死因子-α(TNF-α)的质量浓度.结果 培养的细胞生长状态良好,呈梭形,CD105+细胞占96.8％,CD34+和CD45+细胞分别占2.1％和3.5％.正常对照组眼前节、眼后节检查及视网膜组织病理学检查未发现异常.与模型对照组比较,BMSCs干预组和TA干预组兔眼低炎症评分的眼数明显增加,差异均有统计学意义(X2=7.25,P=0.01;X2=7.37,P=0.01).眼科B型超声检查可见,BMSCs干预组和TA干预组兔眼玻璃体中点状、团状高回声反射物明显少于模型对照组;光学显微镜下发现,与模型对照组比较,BMSCs干预组和TA干预组兔视网膜结构损害明显减轻,低组织病理学评分眼数明显增加,而高组织病理学评分的眼数减少,差异均有统计学意义(x2=8.76,P=0.00 ;x2=8.68,P=0.00);模型对照组血清及房水中TNF-α质量浓度明显高于正常对照组,差异均有统计学意义(P＜0.05),而BMSCs干预组和TA干预组兔眼血清及房水中TNF-α质量浓度均明显低于模型对照组,差异均有统计学意义(P＜0.05).BMSCs干预组与TA干预组间兔眼前节炎症评分、视网膜病理学评分的眼数分布以及血清和房水中TNF-α质量浓度的差异均无统计学意义(P＞0.05).结论 BMSCs和TA玻璃体腔内注射均可减轻兔EAU的炎症反应,两种药物的疗效接近.
揹景 葡萄膜炎多由自身免疫反應所緻,以往多應用糖皮質激素和免疫抑製劑進行治療,但存在藥物依賴和眼壓升高的風險及其他不良反應.骨髓間充質榦細胞(BMSCs)在抗炎、抑製新生血管方麵具有較好的作用,且具有低免疫原性和免疫調節功能,但其在葡萄膜炎治療方麵的研究較少. 目的 比較BMSCs和麯安奈德(TA)對兔實驗性自身免疫性葡萄膜炎(EAU)的治療效果.方法 從5隻新西蘭大白兔股骨和脛骨骨髓中分離、培養BMSCs併進行傳代,用流式細胞技術檢測細胞錶麵抗原CD105、CD34和CD45的錶達以鑒定細胞,取第3～4代細胞用于實驗.將32隻新西蘭大白兔採用隨機數字錶法隨機分為正常對照組、模型對照組、BMSCs榦預組和TA榦預組.正常對照組不進行任何處理,其餘3箇組用質量分數2％牛血清蛋白(BSA)靜脈註射和玻璃體腔內註射法建立EAU模型.造模成功後24 h,模型對照組經右眼玻璃體腔內註射0.1 ml PBS; BMSCs榦預組以同樣方法註射0.1 ml BMSCs懸液,細胞密度為2×106箇/ml;TA榦預組註射0.1 ml(4 mg)TA.于眼部註射後15d每隔3天裂隙燈顯微鏡下觀察實驗兔的眼前節錶現,併參照Caspi的標準進行炎癥評分;利用眼底綵色照相法及眼部B型超聲檢查法觀察各組兔眼後節錶現;于眼部註射後30 d處死實驗兔,製備眼組織標本,進行常規組織病理學檢查,參照Caspi的標準進行視網膜病理學評分;于眼部註射後每隔3天取各組兔的房水0.1ml及外週血3 ml,採用ELISA法檢測兔血清及房水中腫瘤壞死因子-α(TNF-α)的質量濃度.結果 培養的細胞生長狀態良好,呈梭形,CD105+細胞佔96.8％,CD34+和CD45+細胞分彆佔2.1％和3.5％.正常對照組眼前節、眼後節檢查及視網膜組織病理學檢查未髮現異常.與模型對照組比較,BMSCs榦預組和TA榦預組兔眼低炎癥評分的眼數明顯增加,差異均有統計學意義(X2=7.25,P=0.01;X2=7.37,P=0.01).眼科B型超聲檢查可見,BMSCs榦預組和TA榦預組兔眼玻璃體中點狀、糰狀高迴聲反射物明顯少于模型對照組;光學顯微鏡下髮現,與模型對照組比較,BMSCs榦預組和TA榦預組兔視網膜結構損害明顯減輕,低組織病理學評分眼數明顯增加,而高組織病理學評分的眼數減少,差異均有統計學意義(x2=8.76,P=0.00 ;x2=8.68,P=0.00);模型對照組血清及房水中TNF-α質量濃度明顯高于正常對照組,差異均有統計學意義(P＜0.05),而BMSCs榦預組和TA榦預組兔眼血清及房水中TNF-α質量濃度均明顯低于模型對照組,差異均有統計學意義(P＜0.05).BMSCs榦預組與TA榦預組間兔眼前節炎癥評分、視網膜病理學評分的眼數分佈以及血清和房水中TNF-α質量濃度的差異均無統計學意義(P＞0.05).結論 BMSCs和TA玻璃體腔內註射均可減輕兔EAU的炎癥反應,兩種藥物的療效接近.
배경 포도막염다유자신면역반응소치,이왕다응용당피질격소화면역억제제진행치료,단존재약물의뢰화안압승고적풍험급기타불량반응.골수간충질간세포(BMSCs)재항염、억제신생혈관방면구유교호적작용,차구유저면역원성화면역조절공능,단기재포도막염치료방면적연구교소. 목적 비교BMSCs화곡안내덕(TA)대토실험성자신면역성포도막염(EAU)적치료효과.방법 종5지신서란대백토고골화경골골수중분리、배양BMSCs병진행전대,용류식세포기술검측세포표면항원CD105、CD34화CD45적표체이감정세포,취제3～4대세포용우실험.장32지신서란대백토채용수궤수자표법수궤분위정상대조조、모형대조조、BMSCs간예조화TA간예조.정상대조조불진행임하처리,기여3개조용질량분수2％우혈청단백(BSA)정맥주사화파리체강내주사법건립EAU모형.조모성공후24 h,모형대조조경우안파리체강내주사0.1 ml PBS; BMSCs간예조이동양방법주사0.1 ml BMSCs현액,세포밀도위2×106개/ml;TA간예조주사0.1 ml(4 mg)TA.우안부주사후15d매격3천렬극등현미경하관찰실험토적안전절표현,병삼조Caspi적표준진행염증평분;이용안저채색조상법급안부B형초성검사법관찰각조토안후절표현;우안부주사후30 d처사실험토,제비안조직표본,진행상규조직병이학검사,삼조Caspi적표준진행시망막병이학평분;우안부주사후매격3천취각조토적방수0.1ml급외주혈3 ml,채용ELISA법검측토혈청급방수중종류배사인자-α(TNF-α)적질량농도.결과 배양적세포생장상태량호,정사형,CD105+세포점96.8％,CD34+화CD45+세포분별점2.1％화3.5％.정상대조조안전절、안후절검사급시망막조직병이학검사미발현이상.여모형대조조비교,BMSCs간예조화TA간예조토안저염증평분적안수명현증가,차이균유통계학의의(X2=7.25,P=0.01;X2=7.37,P=0.01).안과B형초성검사가견,BMSCs간예조화TA간예조토안파리체중점상、단상고회성반사물명현소우모형대조조;광학현미경하발현,여모형대조조비교,BMSCs간예조화TA간예조토시망막결구손해명현감경,저조직병이학평분안수명현증가,이고조직병이학평분적안수감소,차이균유통계학의의(x2=8.76,P=0.00 ;x2=8.68,P=0.00);모형대조조혈청급방수중TNF-α질량농도명현고우정상대조조,차이균유통계학의의(P＜0.05),이BMSCs간예조화TA간예조토안혈청급방수중TNF-α질량농도균명현저우모형대조조,차이균유통계학의의(P＜0.05).BMSCs간예조여TA간예조간토안전절염증평분、시망막병이학평분적안수분포이급혈청화방수중TNF-α질량농도적차이균무통계학의의(P＞0.05).결론 BMSCs화TA파리체강내주사균가감경토EAU적염증반응,량충약물적료효접근.
Background Uveitis is caused primarily by autoimmune response,and the conventional therapy is the systemic and topical application of glucocorticoid and immunosuppressive agent.However,these therapies exist a variety of complications.Bone marrow mesenchymal stem cells (BMSCs)are verified to have the anti-inflammation and anti-angiogenesis effects with low immunogenicity and immunoregulation function.But little study is reported for its efficacy in treating uveitis.Objective This study was to compare the therapeutic effects of BMSCs and triamcinolone acetonide (TA)on experimental autoimmune uveoretinitis (EAU).Methods BMSCs were isolated and cultured from five New Zealand white rabbits,and the third and fourth generation of cells were used in the experiment.The cells were identified by assay of CD105,CD34 and CD45 using flow cytometry.Thirty-two New Zealand white rabbits were randomized into normal control group,model control group,BMSCs intervention group and TA intervention group according to randomized digital table.EAU models were established by intravenous and intravitreal injection of bovine serum albumin (BSA).After modeling,0.1 ml PBS was intravitreally injected in the model control group,and 0.1 ml BMSCs suspension (2×106/ml)or 0.1 ml (4 mg)TA was used in the same way in the BMSCs intervention group or TA intervention group,respectively.The response of rabbit ocular anterior segment was examined under the slit lamp microscope and scored based on the criteria of Caspi,and the manifestation of ocular posterior segment was evaluated by color fundus photography and B-sonography.The histopathological examination was performed and scored based on the criteria of Caspi 30 days after modeling.Peripheral blood and aqueous humor were obtained for the assay of tumor necrosis factor-α (TNF-α) using ELISA.Results Cultured cells grew well and presented the spindle-like in shape,with the CD105+cells for 96.8％,CD34+cells for 2.1％ and CD45+cells for 3.5％.No obvious abnormality was seen in ocular anterior segment,posterior segment and retinal structure in the normal control group.The number of eyes with the low inflammatory scores was more in the BMSCs intervention group and TA intervention group than that of the model group (x2 =7.25,P =0.01 ;x2 =7.37,P =0.01).The hyperecho reflections were more in the eyes of the model control group compared with the the BMSCs intervention group and TA intervention group on the B-sonography.The damage of the retinal structure could be ameliorated by BMSCs and TA,and the pathological scores of the retinas in the BMSCs intervention group and TA intervention group were significantly lower than those of the model control group (x2 =8.76,P =0.00 ;x2 =8.68,P =0.00).The serum and aqueous TNF-α levels were significantly elevated in the model control group compared with the normal control group (both at P＜ 0.05),and those in the BMSCs intervention group and TA intervention group were significantly declined in comparison with the normal control group (all at P＜0.05).No significant differences were found in the number of eyes of different inflammatory scores,pathological scores of retinas,serum and aqueous TNF-α levels between the BMSCs intervention group and the TA intervention group (all at P＞0.05).Conclusions Intravitreal injection of BMSCs or TA can ameliorate rabbit EAU and BMSCs have a similar therapeutic effect to TA.
