【作者】 韦琦；

【导师】 谭少健；

【作者基本信息】 广西医科大学 ， 眼科学， 2011， 硕士

【摘要】 目的：探讨使用四氧嘧啶(Alloxan, ALX)静脉注射制作糖尿病兔晶状体后囊膜混浊(Posterior capsular opacification, PCO)模型并降低死亡率的方法,并观察高血糖对晶状体PCO形成的早期影响；研究在糖尿病兔PCO发生过程中,去整合素Kistrin对晶状体上皮细胞(Lens epithelial cells, LECs)增殖、细胞缝隙连接蛋白43 (Connexin 43, Cx-43)禾口晶状体后囊膜Ⅳ型胶原蛋白表达的早期影响,为去整合素Kistrin防治糖尿病后发性白内障提供的实验依据。方法：将清洁级健康新西兰雄性大白兔60只随机分为2组；其中30只兔经耳缘静脉一次性注射ALX 90 mg/kg建立糖尿病模型作为高血糖组,另30只兔以同样的方法注射等量生理盐水作为正常血糖组。药物注射后至2周时兔血糖升高到12.0mmol/L以上可判断为建模成功。然后2组分别行兔右眼透明晶状体囊外摘出术(Extracapsular lens extraction surgery, ECLE)。于术后第6、10、14天每组随机选取3只兔进行取材,并对晶状体PCO进行分级。应用免疫组织化学法观察增生细胞核抗原(Proliferating cell nuclear antigen, PCNA)在后囊膜上LECs中的表达情况。然后根据上述实验所确定的PCO形成的时间点,将12只糖尿病兔PCO模型随机分为对照组和实验组,在ECLE术毕时实验组前房内注入80ng/ml的Kistrin溶液0.2ml,对照组注入0.2ml林格氏液,选择在术后第14天时取材,分别采用免疫组化学染色法、免疫荧光技术观察80ng/ml的Kistrin对糖尿病兔LECs中PCNA、晶状体后囊膜Ⅳ型胶原以及Cx-43的表达影响。结果：ALX注射后糖尿病兔的成模率为70%。术后第6、10、14天时,高血糖组兔体质量均明显低于正常血糖组,但血糖明显高于正常血糖组,差异均有统计学意义(P<0.05)。高血糖组术后14d时2只兔出现后囊膜2级混浊,另1只兔出现1级混浊。正常血糖组术后14d时3只兔后囊膜均出现1级混浊。术后6d、10d及14d的PCO分级在两组间均无统计学差异(P>0.05)。免疫组织化学染色显示,术后第10天高血糖组可见PCNA在LECs的细胞核中表达,但正常血糖组未见PCNA的表达。术后第14天时高血糖组PCNA增生指数(Average proliferative index, PI)为0.78±0.10,明显高于正常血糖组的0.25±0.03,差异有统计学意义(t=-16.171,P=0.000)。在Kistrin干预后实验组的PI值为0.57±0.11,Cx-43荧光表达面积比为(0.016±0.002)%,Ⅳ型胶原的积分光密度(Integrated optical density, IOD)平均值为170.6±8.5；对照组的PI值为0.78±0.10,Cx-43荧光表达面积比为(0.136±0.026)%,Ⅳ型胶原的IOD平均值为255.7±15.6。实验组的各项指标表达均比对照组的明显降低(P<0.05)。结论：90 mg/kg的ALX静脉注射能形成稳定的糖尿病兔PCO模型；高血糖是促进晶状体上皮细胞增殖的重要因素之一。去整合素Kistrin可有效抑制晶状体上皮细胞PCNA、Cx-43及后囊膜Ⅳ型胶原表达,减少PCO的形成。更多还原

【Abstract】 OBJECTIVE:This study was to establish the diabetic rabbit’s posterior capsular opacification (PCO) model by intravenous injection of alloxan (ALX), and to explore the method of reducing mortality and the early effects of high blood glucose on PCO. Besides, the early impact of Kistrin on the proliferation of lens epithelial cells (LECs), connexin43 and the expression of collagen IV was also investigated, which will provide experimental evidence for prevention and treatment of diabetic cataract by using Kistrin.METHORD:Sixty clean healthy male New Zealand white rabbits were randomly divided into 2 groups.90mg/kg of alloxan were injected via ear vein once in 30 rabbits to create the diabetic animal models, and the equivalent amount of normal saline solution was injected at the same way as normal blood glucose group. The successful models were selected in the animals with the blood glucose level over 12.0 mmol/L two weeks later, and PCO of lens were graded based on the method of Odrich under the slit lamp. Extracapsular lens extraction was then performed on the right eye of rabbits in both groups, and the posterior capsules were obtained from these eyes at the 6th,10th and 14th days after operation. The expression of proliferating cell nuclear antigen (PCNA) in posterior capsular lens epithelial cell was detected by immunohistochemistry. According to these results, twelve diabetic rabbit’s PCO models were randomly divided into experimental group and control group,0.2ml of Ringers solution and 0.2ml of 80ng/ml Kistrin were injected into the surgery eyes’anterior chamber of rabbits in control group and experimental group, respectively. The expressions of PCNA, connexin43 and collagen IV in LECs of diabetic rabbits at 14th day after surgery were evaluated in two groups by using immunohistochemical staining and immunofluorescence.RESULT:The modeling successful rate was 70% after injection of alloxan. The body weight of rabbits in high blood glucose group was significantly lowed and the blood glucose was significantly elevated in comparison with normal blood glucose group (all P<0.05). Two weeks after surgery,2 eyes occurred 2 grade of PCO and only one eye showed the 1 grade of PCO in the high blood glucose group. while three rabbits in the normal blood glucose group appeared 1 grade of PCO. There were no statistically significant between the two groups in the grade of PCO at the 6th,10th,14th day after surgery (all P>0.05). Biopsy revealed that PCNA was positively expressed in the cell nuclei of LECs in high blood glucose group rather than the normal blood glucose group from the 10th day after surgery. The proliferation index (PI) of PCNA was 0.78±0.10 and 0.25±0.03 respectively in high blood glucose group and normal blood glucose group, showing a significant difference between them (t=-16.171, P=0.000). The PI index of experimental group and control group were 0.57+0.11, 0.78±0.10. respectively, The ratio of Cx-43 positive fluorescent area was (0.016±0.002)% in the experimental group and (0.136±0.026)% in the control group. The Integrated optical density (IOD) of type IV collagen was 170.6±8.5 in the experimental group and 255.7±15.6 in the control group. All these measurements in the experimental group were statistically significantly lower than those in the control group (all P＜0.05).CONCLUSION:Stable diabetic models of rabbits can be created by intravenous injection of 90 mg/kg ALX. High blood glucose level is one of the important factors for the proliferation of LECs. Kistrin with concentration of 80ng-ml-1 could significantly inhibit the expressions of PCNA, Connexin 43 and collagen IV in LECs of diabetic rabbits at 14 days. Kistrin can effective inhibit the PCO formation in early stage.更多还原