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1、1CTGFsiRNA 改善 STZ 诱导糖尿病大鼠视网膜细胞凋亡作者：杨宏伟，陈晓隆，刘哲丽，刘洁，卜立敏【摘要 】 目的：探讨 CTGF 对糖尿病大鼠视网膜内细胞凋亡的影响。方法：将 60 大鼠分为对照组，糖尿病 4，8，12，16wk组和干预组。糖尿病大鼠应用 STZ 腹腔注射诱导成模。干预组大鼠于糖尿病成模后 16wk 玻璃体腔注射 CTGFsiRNA 来造成 CTGF 基因的沉默。取各组视网膜组织，应用 RT-PCR 检测视网膜内的CTGF 基因表达，Tunnel 法检测视网膜凋亡细胞的情况。结果：糖尿病组视网膜内 CTGF 表达和凋亡细胞数较正常组明显增加。凋亡发生在建模后 4wk，
2、并随着时间的延长而加重，在 24wk 时，凋亡细胞数为 25.8 个/mm2 。CTGF 表达于 8wk 时出现升高，到 16wk时升高更明显。CTGFsiRNA 处理后凋亡细胞数明显降低。视网膜内 CTGF 和细胞凋亡之间具有明显的相关性。结论：CTGF 参与了糖尿病视网膜早期病变的细胞凋亡机制，CTGFsiRNA 有利于改善糖尿病早期视网膜由于凋亡所致的细胞丢失。 【关键词】 凋亡; CTGF; 视网膜; 糖尿病AbstractAIM: To detect the effect of CTGF on the 2apoptosis in the diabetic retina with sm
3、all interfering RNAs (siRNA) targeting with CTGF. METHODS: A total of 60 rats were divided into six groups including control group, diabetic 4,8,12,16 weeks group, and interference group. Diabetic rats were induced by STZ intraperitoneal. At 4, 8, 12, 16 weeks after diabetic setting up, retinas were
4、 obtained from control, diabetic rats and diabetic animals treated by intravitreal injection of CTGFsiRNA to suppress the expression of CTGF mRNA. Retinal cells apoptosis was detected by Tunnel staining and mRNA expression of CTGF was analyzed by RTPCR.RESULTS: The levels of CTGF and the apoptosis i
5、n the retinas of diabetic rats were significantly higher than those in the controls. Apoptosis occurred at 4 weeks after a diabetic model setting up, became serious with the diabetes developing, while CTGF elevated at 8 weeks. The cell apoptosis counts increased to 25.8cells/mm2 at 24 weeks of diabe
6、tes. SiRNAmediated inhibition of CTGF mRNA resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and apoptosis in the retina.CONCLUSION: These results suggest that CTGF might be involved in retinal cells apoptosis which is a characteristic of 3early diabet
7、ic retina. siRNA targeting CTGF seems to have the advantage of ameliorating retinal cells lost.INTRODUCTIONDiabetic retinopathy is one of the most common complications of diabetes and a leading cause of vision loss, ultimately resulting in an advanced stage of proliferative retinopathy with neovascu
8、larization, fibrovascular proliferation and retinal detachment. At the cellular level, diabetes alters the function and structure of all retinal cell types1. The pathogenesis of diabetic retinopathy includes glucosemediated microvascular damage24 and alterations of the neural retina，impaired glial r
9、eactivity and apoptotic cell death of retinal cells have been observed in cases of shortterm experimental diabetes and in humans with diabetes5. Animal studies show accelerated apoptosis of retinal neurons6, glial activation7, microvascular cells8, photoreceptors9 and microglial cell10. It is import
10、ant to characterize the early pathological processes in the diabetic neural retina before the onset of vascular pathology. More recently, a novel, cysteine rich secreted protein CTGF has 4been associated with diabetic retinopathies, which is postulated to have prosclerotic，angiogenic and apoptosis i
11、nduction11 properties12 in other tissues. However, there have been few previous reports that have linked the elevated expression of CTGF in diabetic retinas to pathological changes. To investigate the role of diabetesenhanced CTGF expression in retinal cell apoptosis, diabetic rats were treated with
12、 CTGFsiRNA intravitreal injection.MATERIALS AND METHODSMaterials Wistar rats were purchased from Animal Laboratories of China Medical University. Throughout the study animals were given access to food and water in metabolic cages. All animal procedures were in accordance with guidelines set by the A
13、nimal Experiment Committee of the China Institutes for Biological Sciences. Wistar rats, male, weighing 180200g, were randomly divided into six groups: control group, diabetic groups at 4 weeks (DM4W), 8 weeks (DM8W), 16 weeks (DM16W), 24 weeks (DM24W) and DM16W interfered with CTGFsiRNA group (n=10
14、) .5MethodsStreptozotocininduced diabetic rat model Experimental diabetes was induced by intraperitoneal injection of cell toxin streptozotocin (60mg/kg). Immediately prior to use, streptozotocin was dissolved in cold 0.1mol/L citrate buffer, pH 4.5. Control rats received an injection of 0.1mol/L ci
15、trate buffer alone. Blood glucose (BG) levels were measured before and 72 hours after the STZ injection, urinary glucose (UG) measured consequently the first three days. Only the animals with UG above +,blood glucose levels 16.7mmol/L were considered diabetes. Body weight, UG, BG and glycated hemogl
16、obin were measured weekly. At 4, 8, 16, 24 weeks of diabetes, ten rats were randomly selected from the normal control and diabetic groups and killed with a lethal dose of pentobarbital sodium. Eyes from each rat were rapidly enucleated, one being snapfrozen in liquid nitrogen and stored in 80 for the consequent RTPCR, while the contralateral eye was fixed at 40g/L paraformaldehyde for apoptosis and immunohistochemistry.Table 1The s
