靶向抑制GLUT1对糖尿病视网膜病变的作用研究

发布时间：2018-04-01 20:31

本文选题：葡萄糖转运蛋白-1　切入点：小分子干扰RNA　出处：《南昌大学》2016年博士论文

【摘要】：目的:建立糖尿病视网膜病变小鼠模型,观察通过靶向抑制视网膜上葡萄糖转运蛋白-1(Glucose Transporter-1,GLUT1)对糖尿病小鼠视网膜葡萄糖含量的影响。方法:36只8周大小的C57BL/6小鼠按随机数字表法分成正常对照组、糖尿病对照组和GLUT1小干扰核糖核酸(si RNA)组。腹腔注射链脲佐菌素建立糖尿病模型后,GLUT1si RNA组予以玻璃体腔注射1μL靶向GLUT1的si RNA,正常对照组和糖尿病对照组注射等量非靶向性si RNA。建模21周后3组小鼠行免疫印迹法检查视网膜GLUT1的表达并测定比较三组小鼠视网膜的含糖量。结果:GLUT1在神经视网膜层的表达GLUT1si RNA组较正常对照组表达约下降77.00%,仅为糖尿病对照组的8.07%,差异有统计学意义(P0.01)。尽管糖尿病对照组和GLUT1si RNA组视网膜组织含糖量均高于正常对照组,但GLUT1si RNA组小鼠视网膜含糖量为糖尿病对照组的50.05%(P0.01),差异有统计学意义;结论:GLUT1 si RNA可以有效抑制GLUT1在视网膜中的表达,限制葡萄糖转运进入视网膜,从而降低视网膜含糖量。目的:研究限制葡萄糖转运后对糖尿病小鼠视网膜光感受器退行性变的影响。方法:将前述3组小鼠行闪光视网膜电图检查视杆细胞和视锥细胞的功能,通过免疫荧光共定位法检查视锥细胞的密度及形态改变。行HE染色切片测量距离视乳头上下方0.2mm、04mm、0.6mm、0.8mm、1.0mm、1.2mm、1.4mm、1.6mm、1.8mm处视网膜外核层厚度以检查视杆细胞形态学变化。结果:糖尿病对照组和GLUT1 si RNA组小鼠的暗适应和明适应视网膜电图发现a波及b波振幅均低于正常对照组,但GLUT1 si RNA组较糖尿病对照组暗适应视网膜电图高44.6%和94.9%,明适应视网膜电图高47.59%和42.61%,差异均有显著统计学意义(P0.01);形态学检查分别发现GLUT1 si RNA组和糖尿病对照组小鼠的外核层较正常对照组薄,而GLUT1 si RNA组的外核层较糖尿病对照组小鼠厚,差异有统计学意义(P0.05)。糖尿病对照组较GLUT1 si RNA组其视锥细胞排列更为稀疏,外节形态更为短小。结论:GLUT1 si RNA限制葡萄糖转运进入视网膜,减轻了糖尿病小鼠视网膜视锥细胞和视杆细胞功能和形态学退变,对糖尿病视网膜病变光感受器退行性病变产生缓解作用。目的:研究限制葡萄糖转运后对糖尿病小鼠视网膜微血管病变的影响。方法:将前述3组小鼠取视网膜组织行免疫印迹法检查比较视网膜ICAM-1和TNF-α的表达水平的改变,异硫氰酸荧光素-刀豆蛋白A行心内注射以标记粘滞的白细胞并行视网膜平铺以计数比较视网膜血管白细胞粘滞,异硫氰酸荧光素-牛血清白蛋白行股静脉注射并行视网膜平铺以比较血-视网膜内屏障渗漏程度。结果:糖尿病对照组和GLUT1 si RNA组小鼠视网膜ICAM-1和TNF-α的表达均高于正常对照组(P0.01),但这两个炎症因子在GLUT1 si RNA组表达仅为糖尿病对照组的66.14%(P0.05)和54.76%(P0.01),差异有统计学意义;同时我们发现糖尿病对照组较GLUT1 si RNA组有更多的白细胞粘附在视网膜血管中,视网膜铺片发现糖尿病对照组较GLUT1 si RNA组其荧光渗漏区域较多,且渗漏面积也较大。结论:GLUT1 si RNA限制葡萄糖转运进入视网膜,减轻了糖尿病视网膜的炎症反应和血-视网膜内屏障渗漏,对糖尿病小鼠视网膜微血管病变产生缓解作用。
[Abstract]:Objective: to establish a mouse model of diabetic retinopathy, through the observation of targeted inhibition of retinal glucose transporter -1 (Glucose Transporter-1 GLUT1) effects on retinal glucose levels in diabetic mice. Methods: 36 8 week old C57BL/6 mice were randomly divided into normal control group, diabetic control group and GLUT1 small interfering RNA (Si RNA) group. Intraperitoneal injection of streptozotocin diabetic model, GLUT1si RNA group received intravitreal injection of 1 L RNA targeting Si GLUT1, normal control group and diabetic control group was injected with non targeting Si RNA. after 21 weeks of modeling, 3 mice for Western blot examination of retinal GLUT1 the expression was compared between the three groups of mice retinal sugar. Results: GLUT1 expression in the neural retina layer GLUT1si RNA group compared to the control group the expression decreased about 77%, the control was only diabetes Group 8.07%, the difference was statistically significant (P0.01). Although the diabetic control group and GLUT1si group RNA retinal tissue sugar content were higher than the normal group, but GLUT1si group RNA mouse retina sugar for diabetes control group 50.05% (P0.01), the difference was statistically significant; conclusion: the expression of GLUT1 Si RNA can effectively inhibit GLUT1 in in the retina, limiting glucose transport into the retina, thereby reducing retinal sugar. Objective: To study the limit of glucose transport in diabetic mice after photoreceptor degeneration effect. Methods: the 3 groups of mice by electroretinogram examination of rods and cones function, change the density and morphology of cone test cells by immunofluorescence colocalization. HE staining for measuring distance epipapillary below 0.2mm, 04mm, 0.6mm, 0.8mm, 1.0mm, 1.2mm, 1.4mm, 1.6mm, 1.8mm of retina The thickness of the outer nuclear layer to check as the changes in rod cell morphology. Results: the diabetic control group and GLUT1 Si Group RNA mice scotopic and photopic ERG a wave and b wave amplitude were lower than the normal control group, but the GLUT1 Si RNA group compared with the diabetic control group 44.6% dark adaptation electroretinogram photopic retinal and 94.9%. 47.59% and 42.61% were higher, the differences were statistically significant (P0.01); GLUT1 Si RNA were found in the morphological examination group and diabetes control group in the outer nuclear layer than in the normal control group thin, and the outer nuclear layer of GLUT1 Si in RNA group compared with diabetic control group were thicker, the difference was statistically significant (P0.05) diabetes. Compared with the control group GLUT1 Si Group RNA cones arranged more sparse, shorter form outer segments. Conclusion: GLUT1 Si RNA to limit glucose transport into the retina, reduce the retinal cone cells and diabetic mice Rod cell function and morphological changes, alleviate the effect of diabetic retinopathy caused photoreceptor degenerative diseases. Objective: effects on microangiopathy in diabetic mice retinal glucose transport after the research limitations. Methods: expression level of the 3 groups of mice retinal tissue by immune blotting test comparison of retinal ICAM-1 and TNF- alpha changes fluorescein isothiocyanate, concanavalin A underwent intracardiac injection to mark the viscosity of white blood cell count in parallel each retina retinal vascular leukocyte adhesion, fluorescein isothiocyanate bovine serum albumin were injected into the femoral vein parallel each retina to compare the blood retina barrier leakage degree. Results: the expression of GLUT1 and diabetic control group Si RNA mice retinal ICAM-1 and TNF- alpha were higher than the normal control group (P0.01), but the two GLUT1 Si inflammation in group RNA As only the diabetic control group 66.14% (P0.05) and 54.76% (P0.01), the difference was statistically significant; at the same time, we found that the diabetic control group compared with the GLUT1 Si RNA group had more leukocyte adhesion in the retinal vascular, retinal flatmount diabetic group compared with GLUT1 group RNA si the fluorescence leakage and leakage area more the area is also larger. Conclusion: GLUT1 Si RNA to limit glucose transport into the retina, reduce inflammation and blood retinal barrier leakage in the retina, micro vascular lesions in diabetic mice retina with ease.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R774.1

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