维生素E对链脲菌素所致胰岛β细胞损伤的干预研究

发布时间：2018-01-13 06:01

本文关键词：维生素E对链脲菌素所致胰岛β细胞损伤的干预研究　出处：《重庆医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:探讨维生素E(vitamin E)对链脲菌素(streptozotocin,STZ)所致大鼠离体胰岛β细胞损伤的保护途径及可能的机制。方法:1.分组:分离1-3日龄Wistar大鼠胰腺进行体外培养,培养细胞分为4组:①正常对照组:培养的胰岛细胞不作任何干预处理;②维生素E组:培养液中仅加入终浓度为0.1mM的维生素E作用60分钟;③STZ组:培养液中仅加入终浓度为2.2mM的STZ作用30分钟;④保护组:I-IV组。培养液中分别加入0.0125mM、0.025mM、0.05mM、0.1mM的维生素E预作用60分钟后,再加入终浓度为2.2 mM的STZ作用30分钟。各组作用时间一到,离心弃上清液,再用新鲜培养液重悬细胞,继续培养18小时后进行各项指标检测。2.检测:放射免疫法检测培养上清液中胰岛素含量,分光光度计分别测量培养上清液中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,透射电镜观察细胞形态,Hoechest33258荧光染色观察荧光强度,流式细胞仪定量检测细胞凋亡率,分光光度法测量胰岛细胞特异性半胱氨酸酶-3(caspase-3)活性。结果:与正常对照组比较,维生素E组胰岛素含量、SOD活性、MDA水平、细胞凋亡率及caspase-3活性未见明显变化(p㧐0.05)。单纯STZ作用后,胰岛素含量、SOD活性显著降低,MDA水平、细胞凋亡率及caspase-3活性明显升高,与正常对照组比较,p㩳0.05。同时,电镜观察到凋亡细胞,Hoechest33258荧光染色发现发明亮淡蓝色强荧光细胞数显著增多。而在STZ作用前给予不同浓度维生素E作预处理的四个保护组,胰岛素含量、SOD活性、MDA水平虽未恢复到正常对照组水平,但其下降或上升的程度较STZ组出现明显抑制(p㩳0.05)。而且其抑制程度随维生素E浓度的增大而增强(各保护组间比较p㩳0.05),其中以终浓度为0.1mM的保护作用最强,呈现出一定的剂量依赖性趋势。在终浓度为0.1mM的维生素E保护组,细胞凋亡率、caspase-3活性分别为7.84±1.75%和0.153±0.034,与STZ组比较,差异有显著性(p㩳0.05)。结论:维生素E对于STZ诱导的胰岛β细胞损伤有保护作用,并呈现出一定的剂量依赖性趋势。其机制可能与增加SOD活性,清除氧自由基,减少脂质过氧化,降低MDA水平,从而降低caspase-3活性,减少胰岛细胞凋亡有关,抗氧化性可能是其众多保护胰岛β细胞功能的机制之一。
[Abstract]:Objective: To investigate the protective pathway and possible mechanism of vitamin E (vitamin E) on pancreatic islet beta cell injury induced by streptozotocin (STZ) in rats.
Methods: 1. groups: isolated pancreas of 1-3 day old Wistar rats were cultured in vitro. The cultured cells were divided into 4 groups: normal control group: islet cells cultured without any intervention treatment; the vitamin E group: Cultured only with the final concentration of liquid of vitamin E 0.1mM for 60 minutes; group STZ: the medium only with the final concentration of STZ 2.2mM for 30 minutes; the protection group: I-IV group were cultured with 0.0125mM, 0.025mM, 0.05mM, 0.1mM and vitamin E pretreatment for 60 minutes, then add a final concentration of 30 STZ for 2.2 minutes. Each mM action time to centrifugal discard the supernatant with fresh medium cell suspension cultured for 18 hours after the index to detect the.2. content in the supernatant of culture: insulin was measured by radioimmunoassay and spectrophotometry were measured in supernatants of superoxide dismutase (SOD) activity and malondialdehyde (MDA) The cell morphology was observed by transmission electron microscope. The fluorescence intensity was observed by Hoechest33258 fluorescence staining. The apoptosis rate was quantitatively detected by flow cytometry, and the activity of -3 (caspase-3) was measured by spectrophotometry.
Results: compared with normal control group, vitamin E group insulin content, SOD activity, MDA level, significant changes in cell apoptosis rate and the activity of Caspase-3 was not (P? 0.05). The insulin content in STZ group, and SOD activity decreased significantly, the level of MDA, the apoptosis rate and caspase-3 activity increased significantly, and the normal control group comparison of P? 0.05. were observed at the same time, apoptotic cells, Hoechest33258 staining found a bright blue fluorescence cells increased significantly. While in STZ before treated with different concentrations of vitamin E four pretreatment protecting group, insulin content, SOD activity, MDA levels did not return to the normal level of the control group however, the decrease or increase than the degree of group STZ significantly inhibited (P? 0.05). But the degree of inhibition increased with the increasing concentration of vitamin E (the protection group P? 0.05), the final concentration of 0.1mM's protective effect Strong, showing a certain dose dependent trend. In the vitamin E protection group with final concentration of 0.1mM, the apoptotic rate and caspase-3 activity were 7.84 + 1.75% and 0.153 + 0.034, respectively, compared with STZ group, the difference was significant (P? 0.05).
Conclusion: vitamin E on islet beta cell injury induced by STZ has a protective effect, and showed a dose-dependent trend. Its mechanism may be related to increased SOD activity, scavenging oxygen free radicals, reducing lipid peroxidation, reduce the level of MDA, thereby reducing the activity of Caspase-3, reduce the apoptosis of islet cells, antioxidant activity may be one of the the protection mechanism of beta cell function.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341;R96

【参考文献】