【作者】 左艳霞；

【导师】 卞小芸； 宋秀君；

【作者基本信息】 河北医科大学 ， 眼科学， 2008， 硕士

【摘要】 目的:糖尿病(DM)是以糖代谢紊乱为主的常见全身性疾病,患病率逐年提高。DM可引起眼部多种并发症,近年来的临床资料观察到DM患者出现眼干涩、异物感、烧灼感的症状较普遍。本实验制作链脲佐菌素(STZ)大鼠DM动物模型,通过测量泪膜破裂时间(BUT)和基础泪液分泌量(Schirmer-Ⅰ试验)观察DM对照组(DM组)及氨基胍(AG)治疗组(治疗组)大鼠眼表稳定性的改变;并通过观察泪腺、结膜和角膜的组织病理学改变及肿瘤坏死因子-α(TNF-α)、核转录因子-KBp65(NF-KBp65)、糖基化终产物受体(RAGE)的免疫组织化学染色的改变,探讨DM造成的眼表组织改变、发病机制以及AG对DM眼表病变的治疗效果,从而为DM眼表改变提供病理学基础,并为其治疗DM眼表疾病提供新的线索和理论依据。方法:1实验分组将30只180-200g雄性Wistar大鼠随机分为3组:正常对照组(正常组)10只,DM对照组(DM组)10只,AG治疗组(治疗组)10只。2 DM动物模型的制备禁食12h后,对三组大鼠碘伏消毒腹腔注射部位,DM组和治疗组大鼠腹腔一次性注射10g/L的STZ 65mg/kg(溶于20mmol/L枸橼酸钠缓冲液,pH4.5),之后72h和实验中每4wk检测一次血糖,血糖始终大于16.7mmol/L者纳入实验。正常组腹腔一次性注射等量的20mmol/L枸橼酸钠缓冲液(pH4.5)。3给药方式从腹腔注射STZ 72h开始,对三组大鼠碘伏消毒腹腔注射部位,治疗组给予腹腔注射新鲜配制的0.2%AG(80mg/kg.d,用25mmol/L、pH为7.4的磷酸钠缓冲液配制),DM组和正常组分别给予腹腔注射同等剂量的磷酸钠缓冲液(pH为7.4),1/d。4观察指标每日观察各组大鼠的饮水量、饮食量和尿量。每周使用天平称大鼠体重并记录。注射STZ 72h后和每4周大鼠尾端75%酒精消毒后针刺取血,检测血糖。12wk行BUT和Schirmer-Ⅰ试验,脱颈椎法分别处死大鼠每组各10只,立即摘除泪腺、结膜和角膜,用10g/L中性多聚甲醛固定备HE染色和免疫组织化学染色用。结果:1正常组大鼠体重随时间延长而逐渐增加;DM组大鼠和治疗组大鼠在STZ注射后2-12wk体重明显低于正常组,差异有统计学意义(p<0.05),在各时间点,DM组和治疗组大鼠体重差异无统计学意义(p>0.05)(Fig 1)。2 DM组和治疗组大鼠血糖在注射STZ 72h后明显高于正常组,差异具有统计学意义(P<0.01);治疗组和DM组在各时间点大鼠血糖差异无统计学意义(P>0.05)(Fig 2)。3 BUT和Schirmer-Ⅰ试验值的测定:与正常组比较,DM组降低,有统计学意义(p<0.01),治疗组亦降低,有统计学意义(p<0.05);治疗组较DM组明显升高,有统计学意义(p<0.01)(Table 1)。4泪腺、结膜和角膜组织病理学变化:DM组大鼠泪腺腺泡变小,细胞核固缩、深染,细胞分界不清,结膜及角膜损害,表现为结膜杯状细胞减少,角膜上皮和基质层水肿;治疗组大鼠泪腺、结膜和角膜病变程度较DM组大鼠减轻,但较正常组大鼠有明显的差异(Fig 3-11)。5 TNF-α、NF-KBp65和RAGE在各组大鼠泪腺、结膜和角膜的变化:TNF-α、NF-KBp65和RAGE在各组大鼠泪腺、结膜和角膜的胞浆中均有表达;与正常组比较,DM组和治疗组TNF-α、NF-KBp65和RAGE明显升高,差异有统计学意义(p<0.01);治疗组与DM组比较,TNF-α、NF-KBp65和RAGE均降低,差异有统计学意义(p<0.01)(Fig 12-38,Table 2-4)。且在DM组TNF-α、NF-KBp65和RAGE三者之间互相呈正相关关系(Table 5)。结论:1糖尿病与干眼有关。2糖尿病眼表病变与蛋白质糖基化反应有关。3氨基胍对糖尿病引起的眼表病变有一定的治疗作用,其作用机制与影响蛋白质糖基化反应,降低TNF-α、NF-KBp65和RAGE的含量有关;且氨基胍对血糖和体重无影响。更多还原

【Abstract】 Objective:Diabetic mellitus is a common general disease mainly manifestating by glycometabolism disorders whose prevalence is rising year by year. It can cause many complications in eyes. The recent clinical data indicates that the symptom of Dryness of eye, foreign body sensation and burning sensation appeare widespread in DM patients. The experimental model of diabetic rats are induced by intravenous administration of streptozotocin, then we observe the change of ocular surface by Schirmer-Ⅰtest and tear film break up time(BUT) measurement; The histopathological changes and the expressions of tumor necrosis factor alpha(TNF-α), nuclear factor-KBp65(NF-KBp65), receptor for advanced glycation end products(RAGE)of the cornea, conjunctiva and lacrimal gland are observed in the diabetic rats, then we conform diabetic mellitus, the effect of aminoguanidine on it, in order to provide pathological and theoretical basis for treating diabetic ocular surface diseases.Methods:1 Method of grouping30 male Wistar rats that weighted 180-200g,were randomly divided into 3 groups: normal control group (normal group) , diabetic control group (diabetic group) and aminoguanidine treated group (treated group).2 Preparation of diabetic animal modelAfter fasting 12h, three groups’rats were sterilized the site of intraperitoneal injection with Iodophors.Then rats of diabetic and treated group received a single 65mg/kg intraperitonea injection of 10g/L streptozotocin(STZ) dissolved in 20mmol/L sodium citrate buffer (pH 4.5). Detect blood glucose by 72h and per 4 weeks of experiment after STZ injection, then animals that blood levels more than 16.7mmol/L all the time were selected for study. In addition, 10 normal group rats received 20mmol/L sodium citrate buffer (pH 4.5).3 Ways of administration72h after STZ injection, three groups’rats were sterilized the site of intraperitoneal injection with Iodophors. Then treated group received injection of a freshly 80mg/kg.d solution of 0.2% aminoguanidine (25mmol/L phosphate buffered solution, pH7.4) once a day. In addition, diabetic and normal groups received 25mmol/L phosphate buffered solution (pH7.4) alone.4 Indexes to be observedThe volume of drinking, appetite and the urinary volume were observed every day. Body weights of rats were measured weekly. Every 4 weeks during the experiment and 72h after STZ injection, blood glucose of rats were detected. All rats took the Schirmer-Ⅰtest and BUT on the 12th week. Then 10 rats of each group were sacrificed, cornea, conjunctiva and lacrimal gland were obtained and fixed by 10g/L formalin for histopathology and immunohistochemistry.Results:1 Body weight of normal group rats increased gradually. 2-12wk after injection of STZ, the weight was significantly decreased in diabetic and treated groups compared with normal group (p<0.05). Through all facets of the study, the difference of weight beween diabetic and treated groups rats were non-significant(P>0.05) (Fig 1).2 Blood glucose was significantly higher in diabetic and treated groups 72h after injection of STZ,compared with control group (p<0.01); Through all facets of the study, the difference of blood glucose between diabetic and treated groups rats were non-significant(P>0.05)(Fig 2).3 The test values of BUT and Schirmer-Ⅰ: compared with normal group,they were significantly lower in diabetic group(p<0.01),still significantly lower in treated group(p< 0.05); compared with diabetic group, they were significantly greater in treated group(p<0.01) (Table 1).4 Histopathological changes of lacrimal gland, conjunctiva and cornea: Lacrimal glandular follieles of diabetic group shrinked, caryon were pyknosised and anachromasised. Fibers were prolifered, tissues were confused.Goblet cell lost and the basal epithelial cells and the stroma of diabetic groups’cornea were swollen. All of the changes were significantly lightener in treated group than in diabetic group, but still differed from normal group (Fig 3-11).5 Expressions of TNF-α, NF-KBp65 and RAGE in lacrimal gland、conjunctiva and cornea: TNF-α, NF-KBp65 and RAGE are all expressed in the kytoplasm of lacrimal gland、conjunctiva and cornea. Compared with normal group, expressions of TNF-α,NF-KBp65 and RAGE were significantly greater in diabetic and treated groups (p<0.01). But compared with diabetic group, they were significantly lower(p<0.01)(Fig 12-20, Table 2-4). In addition, there were positive correlations in TNF-αand NF-KBp65, TNF-αand RAGE, NF-KBp65 and RAGE (Table 5).Conclusions:1 Diabetic mellitus is related with dry eye.2 Diabetic mellitus ocular disease is concerned with protein glycosylation.3 The effect of AG is significant in treating diabetic mellitus ocular diseases, and its mechanism of action is concerned with protein glycosylation and the decrease of TNF-α, NF-KBp65 and RAGE. In addition, it has no influence on body weight and blood glucose.更多还原