氧化应激对糖尿病大鼠Neuritin和外周神经功能及超微结构的影响

发布时间：2018-05-16 21:16

本文选题：氧化应激 + 糖尿病　；参考：《南京医科大学》2015年硕士论文

【摘要】：目的:1、探讨糖尿病模型状态下,氧化应激对血循环及神经组织中Neuritin水平的影响。2、研究血循环及神经组织中Neuritin水平与神经功能的可能关系。方法:1分组、造模及给药雄性SD大鼠适应性喂养1周,随机选择10只为正常对照组,其余大鼠为实验组。实验组大鼠禁食不禁水12h后,予一次性腹腔注射链尿佐菌素(STZ)55mg/kg,72h后剪尾取血,测血糖16.7mmol/L视为糖尿病造模成功,随后随机分为糖尿病组、低剂量硫辛酸、高剂量硫辛酸组,每组10只。硫辛酸组按20mg?kg-1?d-1或40mg?kg-1?d-1腹腔注射;糖尿病组予等量生理盐水腹腔注射,实验期间自由摄食饮水,给药2、4、6周。2坐骨神经传导速度(MNCV)测定参考Obrosova等[1]方法,分别在造模2、4、6周测定各组坐骨神经MNCV。将大鼠用10%的水合氯醛350mg/kg腹腔注射麻醉后固定于实验平板,两个刺激电极分别位于左侧坐骨切迹、内踝后方,记录电极位于左足底部,单脉冲方波刺激,记录两个刺激点间的距离,输入肌电图仪,得出MNCV,重复三次取平均值。3尾神经传导速度(CNCV)测定刺激电极置于离尾部毛迹120mm处(远端),记录电极置于离刺激电极100mm的轴向距离处(近端),参考电极分别置于离刺激电极近端和记录电极远端约20mm处,刺激电极和记录电极中间接地线.神经反应是由5 Hz频率刺激引起的。直肠温度保持在37℃。4神经组织形态结构观察取一小段坐骨神经,用戊二醛液预固定,经固定、包埋、常规制片,再用醋酸铀和柠檬酸铅双染,最后用Tecnai透射电镜观察。5生化指标检测大鼠尾尖取血,FAD葡萄糖脱氢酶法测定血糖;大鼠眼眶静脉取血,离心得到血清,硫代巴比妥酸法测定丙二醛(MDA)含量,羟胺法测定超氧化物歧化酶(SOD)活性,酶联免疫吸附法测定neuritin浓度,均按试剂盒说明进行检测。6检测坐骨神经、背根神经节neuritin蛋白表达水平提取组织总蛋白,BCA法测定蛋白质浓度,取80μg蛋白实验。经电泳、转膜、封闭后,分别4℃敷育neuritin抗体和β-actin抗体过夜,再用相应的HRP偶联的二抗与其反应,加ECL化学发光试剂,采用Image Lab图像分析软件测定和比较各组neuritin蛋白相对表达量。7坐骨神经和背根神经节的鉴定两种组织的蜡块切成5微米的切片,常规步骤脱蜡。将切片用0.01M PBS处理进行抗原修复,用1%牛血清白蛋白的PBS封闭,接着用一抗和二体的稀释封闭液孵育,细胞核可用4',6二脒基-2-苯基吲哚(DAPI)进行染色。结果:1、糖尿病大鼠血糖明显高于对照组,而糖尿病组与硫辛酸治疗组间无明显差异。2、与正常对照相比,糖尿病组2周后MDA浓度升高,SOD活力下降,neuritin的含量降低(P0.05),4周后MNCV开始下降(P0.05),坐骨神经出现脱髓鞘病变;与糖尿病对照组相比,硫辛酸组MDA浓度下降,SOD活性升高,neuritin的含量增加,MNCV增加(P0.05)。3、正常组的坐骨神经及背根神经节中neuritin的表达高于糖尿病大鼠,2、4、6周的糖尿病大鼠neuritin的表达呈下降趋势。结论:1、糖尿病大鼠中存在严重的氧化应激和neuritin含量的下降,这些变化出现在外周神经功能和结构异常之前。2、硫辛酸可以改善氧化应激,且成一定的量效关系。3、氧化应激可能直接或者通过下调neuritin表达的相关机制引起糖尿病外周神经的异常。
[Abstract]:Objective: 1, to investigate the effect of oxidative stress on the blood circulation and the Neuritin level in the nerve tissue in the state of diabetes mellitus (.2), and to study the possible relationship between the level of Neuritin and the nerve function in the blood circulation and nerve tissue. Methods: 1 groups, the model and the male SD rats were fed for 1 weeks, and 10 rats were randomly selected as the normal control group, and the other rats were randomly selected. For the experimental group, the rats in the experimental group were fasted for 12h, given a one-time intraperitoneal injection of streptozotocin (STZ) 55mg/kg, 72h after cutting the tail and taking the blood, and measuring blood sugar 16.7mmol/L as a successful model of diabetes, then randomly divided into diabetes group, low dose lipoic acid, high dose of lipoic acid group, 10 rats in each group, 20mg kg-1? D-1 or 40mg? Kg-1? D-1 abdomen in the group of lipoic acid. Intraperitoneal injection of the diabetes group was injected with equal amount of normal saline. During the experiment, drinking water was free, and the [1] method of.2 sciatic nerve conduction velocity (MNCV) for 2,4,6 weeks was measured, and the [1] method of Obrosova was measured at 2,4,6 weeks in the model, and the rats in each group of sciatic nerve MNCV. were injected with 10% chloral chloral 350mg/kg in the abdominal cavity and fixed on the experimental plate after the intraperitoneal injection of 10% chloral chloral 350mg/kg. The two stimulation electrodes were located at the left isch incisor and the posterior medial malleolus respectively. The recording electrode was located at the bottom of the left foot, stimulated by single pulse square wave, and recorded the distance between the two stimuli. The electromyogram was entered, and MNCV was obtained, and the three times of the average.3 tail nerve conduction velocity (CNCV) was placed at 120mm (Yuan Duan) and recorded at the tail of the tail. The axial distance (proximal) was placed at the axial distance from the stimulation electrode 100mm. The reference electrodes were placed at the proximal end of the electrode and the distal end of the recording electrode at about 20mm. The stimulation electrode and the recording electrode were grounded. The nerve reaction was caused by the 5 Hz frequency stimulation. The rectal temperature kept a small segment of the sciatic God at the morphological structure of the.4 nerve tissue at 37 degrees centigrade. It was prefixed with glutaraldehyde solution, immobilized, embedded, conventional and double stained with uranium acetate and lead citrate. At the end, blood was detected by.5 biochemical indexes by Tecnai transmission electron microscopy, and blood glucose was measured by FAD glucose dehydrogenase, blood from orbital veins of rats, centrifugation, and determination of malondialdehyde (MDA) by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was measured by hydroxylamine method and the concentration of Neuritin was measured by enzyme linked immunosorbent assay (ELISA). All the sciatic nerves were detected by.6 in the reagent box. The total protein of Neuritin protein in the dorsal root ganglion was extracted. The protein concentration was measured by BCA method and 80 micron protein experiment was taken. After electrophoresis, membrane transfer and closure, it was applied at 4 C respectively. Neuritin antibody and beta -actin antibody were overnight, and the corresponding HRP coupled two antibodies were reacted with ECL chemiluminescence reagents. The Image Lab image analysis software was used to determine and compare the relative expression of Neuritin protein in each group.7 sciatic nerve and dorsal root ganglion. The wax blocks of the two tissues were cut into 5 microns, and the conventional steps were dewaxed. The slice was treated with 0.01M PBS for antigen repair, closed with PBS of 1% bovine serum albumin, and incubated with one and two body diluent closed liquid. The nucleus could be stained with 4', 6 two amid -2- phenyl indole (DAPI). Results: 1, diabetic rats were significantly higher than the control group, and there was no significant difference between the diabetic group and the lipoic acid group.2, Compared with the normal control, the concentration of MDA increased after 2 weeks, the activity of SOD decreased and the content of Neuritin decreased (P0.05). After 4 weeks, MNCV began to decrease (P0.05), and the sciatic nerve appeared demyelinating disease. Compared with the diabetes control group, the concentration of MDA in the lipoic acid group decreased, the SOD activity increased, the Neuritin content increased, MNCV increased (P0.05).3, normal group. The expression of Neuritin in the sciatic and dorsal root ganglia was higher than that in diabetic rats. The expression of Neuritin in diabetic rats was decreased in 2,4,6 weeks. Conclusion: 1, there are severe oxidative stress and a decrease in Neuritin content in diabetic rats. These changes appear before peripheral nerve function and structural abnormalities.2, and lipoic acid can improve oxygen. Oxidative stress and a certain dose effect relationship.3, oxidative stress may directly or through the down-regulation of Neuritin expression mechanism related to diabetic peripheral nerve abnormalities.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【相似文献】