【摘要】目的：探讨二硫苏糖醇（dithiothreitol，DTT）及DTT复合物(含阿司匹林（Asp）、还原型谷胱甘肽（GSH）、赖氨酸（Lys）对SD大鼠硒性白内障的抑制作用。方法：对出生后12d的SD乳鼠（约25g）行皮下注射Na2SeO3（0.2mL/次，隔天注射1次，共计3次，累计剂量约210μg），注射开始后6d（鼠龄18d左右）90%以上的大鼠晶状体发生核性白内障。造模成功后将SD大鼠乳鼠随机分成4组：对照组（C，单独一窝或者是同窝鼠的左眼）、用药组一（D）、用药组二（DAGL）、用药组三（AGL）。分别于造模完成后的第7d给药。C组对照生理盐水点眼；D组25mmol/L的DTT生理盐水溶液点眼；DAGL组DTT复合物（DTT 25mmol/L、Asp 5mmol/L、GSH 2.5mmol/L、Lys 5mmol/L、生理盐水）点眼；AGL组给予不含DTT之外的其他药物成分的生理盐水溶液（Asp 5mmol/L、GSH 2.5mmol/L、Lys 5mmol/L）点眼。5次/d，1滴/次（约25μL），连续3wk。结果：3d内核性混浊均增大，右眼（用药组）<左眼(对照组)；3～4d混浊斑块达峰值，大鼠左右眼斑块直径呈现显著差异（P＜0.01)；此后斑块呈密集浓缩趋势，直径均轻度下降；14d混浊斑块密度增加达峰值，左右眼斑块直径仍呈现显著差异（P＜0.01)。20d右眼发展到全白内障（Ⅵ级）的只数<左眼（P＜0.01)；D、DAGL、AGL组均可抑制SD大鼠硒性白内障的发展，与对照组相比差异显著（P＜0.01)；D、DAGL组对SD大鼠硒性白内障的抑制作用相近（P＞0.05）；D组和DAGL组对SD大鼠硒性白内障的抑制作用均优于AGL组（P＜0.01)。
结论：含DTT的抗白内障药物复合物抑制SD大鼠硒性白内障发展的作用，显著强于不含DTT的其它类抗白内障药物组合。
【关键词】 SD大鼠；硒性白内障；DTT
Comparison the effect of dithiothreitol and its complex on sodiumseleniteinduced cataract of SD neonate rats
Qing Fu, Yu Geng, JianRong Zhang, BoLin Xie, YaJun Peng, Lin Liu, WenJie Li
Foundation item: Key Program of the Chinese PLA during the 11th FiveYear Plan Period (No. 06MB213 )
1Department of Ophthalmology;2Department of Laboratory Medicine, Changhai Hospital of the Second Military Medical University, Shanghai 200433, China; 3 Department of Ophthalmology, General Hospital of Kunming, Chengdu Military District, Kunming 650032, Yunnan Province, China
Correspondence to: WenJie Li. Department of Laboratory Medicine, Changhai Hospital of the Second Military Medical University, Shanghai 200433, China. [email protected]
Abstract AIM: To compare the inhibition effect of dithiothreitol (DTT) and DTT complex (including aspirin (Asp), glutathione (GSH), lysine (Lys)) on SD neonate rats sodiumseleniteinduced cataract. METHODS: Twelveday old SD neonate rats (about 25g) were performed subcutaneous injection of Na2SeO3 (0.2mL/time, every other day, totally three times, cumulative dose was about 210μg). Six days after the injection began (around 18day old rat), more than 90 percent of the lens showed nuclear cataract. SD neonate rats were divided into four groups: the control group (C, selected brood or the left eye in a brood), DTT treatment group 1 (D), the treatment group 2 (DAGL), the treatment group 3 (AGL). Eye drops were presented 7 days after the SD neonate rats cataract were constructed. Eyes in group C were treated by saline eye drops, group D by 25mmol/L DTT dissolved in saline, group DAGL by DTT complex (DTT 25mmol/L, Asp 5mmol/L, GSH 2。5mmol/L, Lys 5mmol/L dissolved in saline solution), and group AGL by dissolution excepted of DTT (Asp 5mmol/L, GSH 2.5mmol/L, Lys 5mmol/L). The eye drops were instilled into the conjunctiva five times a day, one drop every time (about 25μL), for 3 consecutive weeks. RESULTS: On the 3rd day, nuclear turbidity increased, the right eye was smaller than the left eye. On the 34th day, muddy patch reach peak, the diameter of right eye and the left eye showed significant difference (P<0.01). There was a plaqueenrichment trend with the turbidity diameter decline mild. On the 14th day, reach density peak and the diameter eye patch still showed significant differences (P<0.01). After 20 days, the number of whole cataract in the right eye (nuclear turbidity Ⅵ level) was less than that in the left eye. The difference had statistical significance (P<0.01). Compared with control group, inhibition of SD neonate rats cataract was more obvious in D, DAGL, and AGL group (P<0.01). Similar inhibition effect appeared in D and DAGL group (P>0。05), and that in D and DAGL group were better than AGL group (P<0.01).CONCLUSION: DTT contained the eye drops inhibition on the development of the SD neonate rats sodiumseleniteinduced cataract significantly stronger than the combination that the DTT not contained.
KEYWORDS: SD neonate rats; sodiumseleniteinduced cataract; DTT
0引言
DTT是一种强还原剂，有抗氧化作用。为白色针状结晶，熔点42～43℃，沸点125～130℃(0.27kPa)，37℃，0。67Pa时升华，微吸潮，易溶于水、乙醇、丙酮、乙酸乙酯、氯仿和乙醚，在空气中缓慢氧化；以1，4丁二烯为原料，经溴化、氧化、乙酰化后与硫代乙酸钾反应后经甲醇氯化氢溶液水解而得；常用作蛋白质巯基保护剂。体外实验发现DTT可以有效抑制β B2受热聚集变性，减少β晶状体蛋白巯基氧化交联作用，从理论上可以抑制晶状体高分子量蛋白质的形成，具有直接而明确的晶状体蛋白保护作用[1]。目前临床尚缺乏有效抗白内障药物，白内障最终需手术治疗。因此有必要深入探讨白内障的发病机制，探寻能够有效防护和早期预防治疗白内障的药物。本实验尝试用含DTT成分的药物组合对硒性白内障SD大鼠进行干预，取得令人振奋的效果。
1材料和方法
1.1材料
模型建立：对出生后12d的SD乳鼠（约25g）行皮下注射Na2SeO3（0.2mL/次，隔天注射一次，共计3次，累计剂量约210μg），注射开始后6d（鼠龄18d左右）90%以上的大鼠晶状体发生核性白内障[2]。试剂配制：抗白内障药物分别以D、DAGL、AGL表示。各药物成分的摩尔浓度：DTT 25mmol/L，Asp 5mmol/L，GSH 2.5mmol/L，Lys 5mmol/L。置于EP离心管中，4℃冰箱保存，1wk内用完。D：1mol/L（DTT）储存液取5g的DTT加32.4mL水，分成小份贮存于4℃；取0.5mL储存液用生理盐水稀释至20mL，配制成25mmol/L的应用液，pH值调整到6.0。DAGL：取25mmol/L DTT生理盐水溶液，分析天平称取阿司匹林18mg，还原型谷胱甘肽15.3mg，赖氨酸14.62mg，完全溶解于上述溶液中， pH值调整到6.0，置于4℃冰箱保存。 AGL：分析天平称取阿司匹林18mg，还原型谷胱甘肽15。3mg，赖氨酸14.62mg，用20mL生理盐水完全溶解，pH值调整到6.0。
1.2方法
造模成功后，将SD大鼠乳鼠随机分成4组：对照组（C，单独一窝大鼠或者是同窝鼠的左眼）、用药组一（D）、用药组二（DAGL）、用药组三（AGL），分别用苦味酸作背部标记。造模成功后于第7d给药（滴眼）。每次分别用注射器抽取相应的抗白内障药物2mL，给相应组别的SD大鼠滴眼，1滴/(次·眼)。实验中滴入鼠眼的药物不能立刻完全吸收，部分眼药会在乳鼠瞬目时溢出结膜囊；同时SD乳鼠会自行搔挠、清理眼部的部分眼药，这样会减少药物在鼠眼停留的时间，并降低药物在鼠眼结膜囊内的局部浓度，故在每次给鼠眼滴入眼药后，需人工固定鼠眼睑并限制乳鼠的自行搔挠，维持5min左右。每天9∶00,11∶00,15∶00,17∶00,20∶00时给药，维持3wk。分别于造模完成后的第6d（用药前），用药后第5,10,15,20d，用裂隙灯显微镜观察，并按文献分级[3,4]进行记录，同时行裂隙灯照相(×16)。C、D、DAGL、AGL组拍照结果如图1。
统计学处理：晶状体伴发皮质混浊阳性率比较采用组间非配对χ2检验；所有数据均采用SPSS 10.0软件包进行统计学处理。
2结果
用药干预后，在实验观察的时间内（3wk），D、DAGL和AGL组发展到全白内障（Ⅵ级）的眼数均低于对照组，差异显著（P＜0.01）；D组和DAGL组发展到全白内障（Ⅵ级）的眼数明显低于C组和AGL组，均差异显著（P<0.01）；而D组和DAGL组大多数鼠眼停留在核性白内障（Ⅴ级），无显著差异（P＞0.05）。用药后第5d时，对照组SD乳鼠10只（20眼）之双眼形成典型的核性白内障，之后白内障程度呈发展趋势，核性混浊区域逐渐增大，直径增加。 而用药组晶状体混浊程度明显低于对照组，尤其以含有DTT组最明显（表1）；第20d时，疗效更加显著（表2）。D组:SD乳鼠12只（24眼）形成典型的核性白内障。用药后第5d，核性混浊继续发展，但程度和速度较对照组慢。DAGL组:SD乳鼠11只（22眼）形成典型的核性白内障。用药后第5d，核性混浊继续发展，但程度和速度较对照组慢，干预效果与D组无差异。AGL组:SD乳鼠13只（26眼）形成典型的核性白内障。用药后第5d，核性混浊继续发展，其程度和速度较对照组慢，但较D和DAGL组快。表1用药第5d时SD乳鼠晶状体混浊度分级计数眼（略）表2 用药第20d时SD乳鼠晶状体混浊度分级计数眼（略）
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