【作者】 张瑜；

【导师】 叶纹； 徐国彤；

【作者基本信息】 复旦大学 ， 眼科学， 2009， 博士

【摘要】 目的:观察exendin-4类似物(Exendin-4 analogue,E4a)对链脲佐菌素(STZ)造模大鼠的血糖和视网膜形态学、功能学的影响。探讨Exendin-4类似物参与糖尿病大鼠视网膜病变(Diabetic Retinopathy,DR)的保护作用的相关机理。方法:1.建立糖尿病大鼠模型,皮下及玻璃体腔注射exendin-4类似物,化学法观察其对大鼠血糖的影响及对视网膜形态学的影响。反转录聚合酶链式反应(Reverse Transcriptase-Polymerase Chain Reaction,RT-PCR)、蛋白免疫印迹(Western immunoblotting,WB)、免疫组织化学法(Immunohistochemistry,IHC)观察糖尿病大鼠视网膜及其他组织胰升糖素样肽-1受体(Glucagon-like peptide-1 receptor,GLP-1R)的表达情况。2.观察皮下注射exendin-4类似物对糖尿病大鼠视网膜厚度、细胞计数、视网膜电图(Electroretinogram,ERG)的影响。3.观察玻璃体腔内注射exendin-4类似物对糖尿病性大鼠视网膜厚度、细胞计数、视网膜电图的影响。4.观察玻璃体腔内注射E4a后视网膜谷氨酸含量的变化;免疫组化、蛋白免疫印迹、反转录聚合酶链式反应观察糖尿病大鼠视网膜谷氨酰胺合成酶(glutamine synthetase,GS)、谷氨酸转运蛋白(glutamine-aspartatetransporters,GLAST)的表达情况;高糖培养视网膜M(u|¨)ller细胞株,观察其谷氨酰胺合成酶、谷氨酸转运体的表达情况。结果:1,皮下注射Exendin-4类似物0.05μg/g BW/day能取得较好的降血糖效果,玻璃体腔内注射Exendin-4类似物0.1μg对糖尿病引起的视网膜形态学改变的保护最明显。糖尿病大鼠视网膜及其他组织如肝、睾丸等在RNA及蛋白水平均有GLP-1R的表达。2.糖尿病可致大鼠视网膜厚度减小,各层细胞数减少、视网膜电图的振幅降低,而皮下注射E4a可使其受病变影响的程度显著减小。3.玻璃体腔内注射E4a可使糖尿病所致的视网膜变薄、细胞数减少、视网膜电图振幅降低明显改善;4.玻璃体腔内注射E4a后视网膜内谷氨酸含量明显降低;糖尿病大鼠视网膜均有GLP-1R、GLAST的表达,E4a可使其表达上调;高糖培养的M(u|¨)ller细胞株上GLP-1R、GLAST表达减弱,E4a可使其表达上调。结论:1.皮下注射E4a可使糖尿病大鼠的血糖降低,玻璃体腔内注射E4a对糖尿病性视网膜病变具有保护作用。大鼠视网膜、肝、睾丸等组织均有GLP-1R的表达。2.皮下及玻璃体腔内注射E4a具有干预糖尿病对视网膜神经元等组织细胞的损伤,防止其发生凋亡的作用;这种作用可能是通过上调谷氨酸转运蛋白、GLP-1R从而降低视网膜内谷氨酸含量,减小其兴奋毒性作用来完成的。玻璃体腔内注射E4a可能会成为一种治疗早期糖网病、甚至其他视网膜疾病的新方法。更多还原

【Abstract】 Purpose To explore the protective effect of exendin-4 analogue(E4a) on early DR. Methods(1).Exendin-4 analogue was injected subcutaneous(sc.) and intravitreally with different doses to find the best one of protecting the diabetic retinopathy. Expression of GLP-1R was detected at both mRNA and protein levels and verified by immunohistochemistry.(2).Thirty six Sprague-Dawley(SD) rats were included in the experiment,Diabetes was induced by intraperitoneal injection(ip) of streptozotocin (STZ).The rats were divided into 3 groups:normal control(N),diabetic control(D) and E4a-treated diabetic(E4a) group.For E4a group,the rats were treated with E4a (sc.0.05μg/g BW/day);for N and D groups,the rats were treated with normal saline (NS,sc),accordingly.Blood glucose levels and body weight were measured weekly. Electroretinogram(ERG) was performed 1 and 3 months after diabetes onset.The retinal thickness and cell counts in each layer were evaluated under light microscopy after ERG examination.(3).Experimental diabetic rats were injected E4a intravitreally.Electroretinogram(ERG) was performed 1 month after diabetes onset. The retinal thickness and cell counts in each layer were evaluated under light microscopy after ERG examination.The concentration of glutamate in vitreous was tested by HPLC.(4).Expression of GS and GLAST were detected at both mRNA and protein levels and verified by immunohistochemistry.Retinal M(u|¨)ller cell was cultured in medium with high-glucose,and Gene expression of the GS and GLAST were studied with RT-PCR.Western blotting assay was performed to explore the level of GLAST on cell total proteins.Results(1).E4a can reduce the blood glucose level of diabetic rats to the normal control level when it was injected subcutaneously.GLP-1R was expressed at both mRNA and protein levels in retina of SD rats.Immunostaining of the rat retina revealed that GLP-1R was predominantly expressed in inner layer of the retina.(2). B-wave amplitudes and OPs decreased with the progress of diabetes,and E4a injected whether subcutaneous(sc.) or intravitreally prevent the loss of b-wave amplitude and OPs caused by diabetic rats.The retinal thickness was reduced with a diabetes-duration dependent fashion.The cell counts of both ONL and INL were reduced accordingly in the diabetic rats.E4a prevented the cell loss and maintained a normal thickness.The concentration of glutamate in vitreous in E4a injected intravitreally group was significantly reduced compared with the diabetic control.(3). GS and GLAST were expressed at mRNA level in retina of SD rats and M(u|¨)ller cells. Immunostaining of the rat retina revealed that GLAST was predominantly expressed in M(u|¨)ller cells and RGC cells.The protein level of GLAST in E4a group expressed more than diabetic control.Conclusions GLP-1R was expressed in rat retina.Apoptosis is an important constituent of retinal cell death in early DR.E4a administration can reverse the changes of ERG,prevent the retinal cell death and maintain normal retinal thickness in diabetic rats.It can reduce the concentration of glutamate in retina through upregulating the expression of GLAST.Therefore,this is a potent approach for treatment of early DR.更多还原