去整合素Echistatin对糖尿病兔早期后发性白内障的抑制作用及眼内安全性
거정합소Echistatin대당뇨병토조기후발성백내장적억제작용급안내안전성
Effect and safety of different concentrations of disintegrin Echistatin to early posterior capsule opacification in diabetic rabbits
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2014年 11期 975-979 ,共5页

    钱光霞%谭少健%梁皓%陈迎迎

    전광하%담소건%량호%진영영

    去整合素%后发性白内障%增生细胞核抗原%凋亡%糖尿病%动物模型 去整閤素%後髮性白內障%增生細胞覈抗原%凋亡%糖尿病%動物模型
    거정합소%후발성백내장%증생세포핵항원%조망%당뇨병%동물모형
    Disintegrin%Posterior capsular opacification%Proliferating cell nuclear antigen%Apoptosis%Diabetes mellitus%Animal model
    背景 后囊膜混浊(PCO)是影响白内障术后视力的主要原因,糖尿病患者白内障术后更容易发生PCO.研究证实,去整合素Echistatin(Ecs)能抑制体外晶状体上皮细胞(LECs)的增生,从而抑制PCO的形成,但其机制和安全剂量有待证实.目的 研究不同质量浓度Ecs对糖尿病兔早期PCO发生过程中LECs增生的抑制作用及Ecs在眼内应用的安全剂量.方法 用随机数字表法将15只新西兰大白兔分为5个组,用四氧嘧啶兔耳缘静脉注射法建立兔糖尿病模型,然后兔右眼行常规晶状体囊外摘出术,术后单纯糖尿病兔前房注入蒸馏水0.2 ml作为对照组,术后按照分组前房内分别注入5.0、7.5、10.0和15.0 μg/ml的Ecs0.2 ml.术后裂隙灯生物显微镜下观察眼前段炎症反应及PCO形成和分级情况.术后10d取出兔右眼,制备常规石蜡切片,采用免疫组织化学法检测PCO发生过程中LECs中增生细胞核抗原(PCNA)的表达以确定Ecs对PCO抑制的有效剂量;采用常规组织病理学方法检查角膜和视网膜结构的形态学改变,并采用TUNEL法检测角膜内皮细胞及视网膜内核层细胞的凋亡情况,以确定Ecs对眼内组织的安全剂量.结果 各组兔实验眼晶状体囊外摘出术后出现不同程度的角膜水肿及前房渗出,对照组和5.0 μg/ml Ecs组术后3～5 d炎症反应消失,≥7.5 μg/ml Ecs组术眼均于术后7d恢复正常.术后对照组和5.0 μg/ml Ecs组PCO均为1～2级,≥7.5 μg/ml Ecs组术眼PCO均为0级.对照组、5.0 μg/ml Ecs组、7.5 μg/ml Ecs组和10.0 μg/ml Ecs组LECs中PCNA阳性细胞表达值(A值)的总体比较差异有统计学意义(F=18.006,P=0.001),与对照组比较,7.5和10.0 μg/ml Ecs组LECs中PCNA阳性细胞表达值明显下降,差异均有统计学意义(P=0.010、0.001).各组兔眼角膜内皮细胞排列整齐,视网膜内界膜与各层组织结构完整.TUNEL法检测可见≤10.0 μg/ml Ecs各组与对照组间角膜内皮细胞及视网膜内核层细胞凋亡值(A)的差异均无统计学意义(均P＞0.05),而15.0 μg/ml Ecs组较≤10.0μg/ml Ecs各组角膜内皮细胞和视网膜内核层细胞的凋亡值均明显增加,与对照组比较差异有统计学意义(P=0.004、0.018).结论 Ecs对糖尿病兔早期PCO具有抑制作用,其剂量7.5和10 μg/ml时疗效较好且对眼内组织无明显毒性作用,可继续用于其对糖尿病兔PCO抑制的远期效果观察.
    배경 후낭막혼탁(PCO)시영향백내장술후시력적주요원인,당뇨병환자백내장술후경용역발생PCO.연구증실,거정합소Echistatin(Ecs)능억제체외정상체상피세포(LECs)적증생,종이억제PCO적형성,단기궤제화안전제량유대증실.목적 연구불동질량농도Ecs대당뇨병토조기PCO발생과정중LECs증생적억제작용급Ecs재안내응용적안전제량.방법 용수궤수자표법장15지신서란대백토분위5개조,용사양밀정토이연정맥주사법건립토당뇨병모형,연후토우안행상규정상체낭외적출술,술후단순당뇨병토전방주입증류수0.2 ml작위대조조,술후안조분조전방내분별주입5.0、7.5、10.0화15.0 μg/ml적Ecs0.2 ml.술후렬극등생물현미경하관찰안전단염증반응급PCO형성화분급정황.술후10d취출토우안,제비상규석사절편,채용면역조직화학법검측PCO발생과정중LECs중증생세포핵항원(PCNA)적표체이학정Ecs대PCO억제적유효제량;채용상규조직병이학방법검사각막화시망막결구적형태학개변,병채용TUNEL법검측각막내피세포급시망막내핵층세포적조망정황,이학정Ecs대안내조직적안전제량.결과 각조토실험안정상체낭외적출술후출현불동정도적각막수종급전방삼출,대조조화5.0 μg/ml Ecs조술후3～5 d염증반응소실,≥7.5 μg/ml Ecs조술안균우술후7d회복정상.술후대조조화5.0 μg/ml Ecs조PCO균위1～2급,≥7.5 μg/ml Ecs조술안PCO균위0급.대조조、5.0 μg/ml Ecs조、7.5 μg/ml Ecs조화10.0 μg/ml Ecs조LECs중PCNA양성세포표체치(A치)적총체비교차이유통계학의의(F=18.006,P=0.001),여대조조비교,7.5화10.0 μg/ml Ecs조LECs중PCNA양성세포표체치명현하강,차이균유통계학의의(P=0.010、0.001).각조토안각막내피세포배렬정제,시망막내계막여각층조직결구완정.TUNEL법검측가견≤10.0 μg/ml Ecs각조여대조조간각막내피세포급시망막내핵층세포조망치(A)적차이균무통계학의의(균P＞0.05),이15.0 μg/ml Ecs조교≤10.0μg/ml Ecs각조각막내피세포화시망막내핵층세포적조망치균명현증가,여대조조비교차이유통계학의의(P=0.004、0.018).결론 Ecs대당뇨병토조기PCO구유억제작용,기제량7.5화10 μg/ml시료효교호차대안내조직무명현독성작용,가계속용우기대당뇨병토PCO억제적원기효과관찰.
    Background Posterior capsular opacification (PCO) is a primary cause of blurred vision after extra-capsular cataract extraction (ECCE),and there is a higher incidence of PCO in the patients with diabetes mellitus.Echistatin (Ecs) can suppress the proliferation of lens epithelial cells (LECs) and thereby inhibit the formation of PCO.However,its mechanism and safe dose deserve to study.Objective The aim of this study was to observe the inhibitory effect of different concentrations of Ecs on LECs proliferation in the early stage of PCO in diabetic rabbits and explore the safe dose of Ecs.Methods Diabetic mellitus was induced by injection of 90 mg/kg alloxan via ear vein in 15 New Zealand white rabbis.ECCE was performed in the right eyes of the rabbits.The rabbits were randomized to the control group and 5.0,7.5,10.0 and 15.0 μg/ml Ecs group according to randomized number table method.Ecs of 0.2 ml in above doses was injected into the anterior chamber after ECCE in different concentrations of Ecs groups,and 0.2 ml distilled water was used in the same way in the only diabetic rabbits as the control group.Postopeartive response of ocular anterior segment was observed and PCO was graded under the slit lamp microscope.The corneal and retinal specimens were prepared 10 days after operation for the assay of preliferative cell nuclear antigen (PCNA) expression in LECs by immunochemistry to evaluate the effective dose of Ecs.Regular histopathological examination was performed,and apoptosis of corneal endothelial cells and retinal cells was detected by TUNEL method to assess the safe concentration of Ecs.Results Different degrees of corneal edema and exudation in anterior chamber were seen in the eyes of different groups.The inflammatory response disappeared 3-5 days in the control group and 5.0 μg/ml Ecs group and 7 days in the ≥7.5 μg/ml Ecs groups.PCO was 1-2 grade in the control group and 5.0 μg/ml Ecs group and 0 grade in the ≥ 7.5 μg/ml Ecs groups.The difference in the positive expression level (absorbance,A) for PCNA in LECs was significantly different among the control group and various Ecs groups (F=18.006,P=0.001),and the positive expression level of PCNA in the ≥ 7.5 μg/ml Ecs groups was markedly reduced in comparison with that in the control group (P =0.010,0.001).Hematoxylin and eosin staining showed an normal morphology and order arrangement in corneal endothelial cells and intact structure in retinal internal limiting membrane in the groups.TUNEL assay revealed that the apoptosis values (mean A value) of corneal endothelial ceils and retinal cells in the ≤ 10.0 μg/ml Ecs groups were not significantly changed in comparison with the control group (all at P＞0.05),but the apoptosis values in the 15.0 μg/ml Ecs group were markedly higher than those in the control group (P=0.004,0.018).Conclusions Ecs can inhibit the early PCO in diabetic rabbits and show the optimal effect at the concentrations of 7.5 and 10.0 μg/ml without visible eytotoxicity to eye other tissues.Therefore,these two doses of Ees might be used for the study of long-term therapeutic effectiveness.
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