GSK-3β介导地塞米松诱导胰岛β细胞凋亡的体外研究
发布时间：2019-02-23 22:41
【摘要】：目的应用地塞米松(DEX)诱导的大鼠胰岛INS-1细胞凋亡模型。探讨糖原合酶激酶-3β(GSK-3β)在糖皮质激素诱导胰岛β细胞凋亡过程中的作用及可能的分子机制,为进一步阐明类固醇性糖尿病的发生机制提供实验依据。方法1选用大鼠胰岛β细胞株INS-1细胞作为研究对象,首先应用MTT法检测不同浓度(0、0.01、0.1、1μM)DEX作用细胞1,2,3d时细胞增殖情况,确定DEX的最佳作用浓度和作用时间;2不同浓度DEX作用INS-1细胞48h后通过台盼蓝染色、Tunel染色和流式细胞术检测细胞死亡情况;3免疫荧光染色方法观察DEX诱导后,INS-1细胞中GSK-3β和p-GSK-3β(Ser9)的蛋白表达;4 0.1μM DEX处理INS-1细胞48h后,采用Real-time PCR方法检测SOD、i NOS、Nox4、NADPH oxidase(p47phox)和GSK-3βm RNA的表达;采用western-blot方法检测GSK-3β、p-GSK-3β(Ser9)、SOD、i NOS和Nox4蛋白的表达;采用ROS试剂盒、Griess法检测ROS及NO的释放水平。5观察加入GSK-3β抑制剂Li Cl后上述各项指标变化。结果DEX(0、0.01、0.1、1μM)作用INS-1细胞1--3天,随着DEX剂量和时间增加,INS-1细胞存活率下降;Tunel染色、流式细胞术证实0.1μΜ浓度的DEX作用INS-1细胞48h时,细胞出现明显的凋亡现象,并且与对照组相比,细胞培养上清中的ROS、NO水平升高,Nox4、p47phox、i NOS m RNA表达上调,SOD m RNA表达下调;i NOS、Nox4蛋白表达水平升高,SOD、p-GSK-3β(Ser9)蛋白表达明显降低,总GSK-3β蛋白表达无明显变化。加入Li Cl后DEX诱导的细胞凋亡率明显下降,ROS、NO水平降低,Nox4、p47phox、i NOS m RNA表达及i NOS、Nox4蛋白表达均明显下调,p-GSK-3β(Ser9)蛋白表达上调,差异均具有统计学意义(P0.05)。结论地塞米松可诱导大鼠胰岛β细胞凋亡,其机制可能与氧化应激反应增强、GSK-3β活性增加有关;抑制GSK-3β活性在一定程度上可减轻地塞米松诱导的细胞凋亡,其机制可能与减轻氧化应激反应有关。
[Abstract]:Objective to induce apoptosis of rat islet INS-1 cells by dexamethasone (DEX). To explore the role of glycogen synthase kinase-3 尾 (GSK-3 尾) in glucocorticoid-induced apoptosis of islet 尾 cells and its possible molecular mechanism, and to provide experimental evidence for further elucidating the mechanism of steroid induced diabetes mellitus. Methods (1) Rat islet 尾 cell line INS-1 cells were selected as the study object. The proliferation of INS-1 cells at different concentrations (0. 01 渭 M) DEX) (0. 01 ~ 0. 1 渭 M) DEX) for 3 days was detected, and the optimal concentration and time of DEX were determined. (2) the death of INS-1 cells was detected by trypan blue staining, Tunel staining and flow cytometry after treatment with different concentrations of DEX for 48 h. (3) the expression of GSK-3 尾 and p-GSK-3 尾 (Ser9) in INS-1 cells induced by DEX was observed by immunofluorescence staining. The expression of SOD,i NOS,Nox4,NADPH oxidase (p47phox) and GSK-3 尾 m RNA in INS-1 cells were detected by Real-time PCR method after treatment with 40.1 渭 M DEX for 48 h. The expression of GSK-3 尾, p-GSK-3 尾 (Ser9), SOD,i NOS and Nox4 were detected by western-blot method, and the release levels of ROS and NO were detected by ROS kit and Griess assay. 5 the changes of the above indexes were observed after adding GSK-3 尾 inhibitor Li Cl. Results the survival rate of INS-1 cells decreased with the increase of DEX dose and time after treatment with DEX (0. 01 ~ 0. 1 渭 M) for 1 to 3 days. Tunel staining and flow cytometry showed that the apoptosis of INS-1 cells was observed at the concentration of 0.1 渭 m DEX for 48 h. Compared with the control group, the level of ROS,NO in the supernatant of cell culture was increased, and the level of Nox4,p47phox, in the supernatant was higher than that in the control group. The expression of i NOS m RNA was up-regulated and the expression of, SOD m RNA was down-regulated. I NOS,Nox4 protein expression increased, SOD,p-GSK-3 尾 (Ser9) protein expression decreased significantly, total GSK-3 尾 protein expression did not change significantly. After the addition of Li Cl, the apoptosis rate induced by DEX was significantly decreased, the level of ROS,NO was decreased, the expression of Nox4,p47phox,i NOS m RNA and I NOS,Nox4 protein were down-regulated, and p-GSK-3 尾 (Ser9) protein expression was up-regulated. The difference was statistically significant (P0.05). Conclusion Dexamethasone can induce apoptosis of islet 尾 cells in rats. The mechanism may be related to the increase of oxidative stress and the activity of GSK-3 尾. Inhibiting the activity of GSK-3 尾 can reduce the apoptosis induced by dexamethasone to some extent, and its mechanism may be related to the reduction of oxidative stress.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
本文编号：2429289
[Abstract]:Objective to induce apoptosis of rat islet INS-1 cells by dexamethasone (DEX). To explore the role of glycogen synthase kinase-3 尾 (GSK-3 尾) in glucocorticoid-induced apoptosis of islet 尾 cells and its possible molecular mechanism, and to provide experimental evidence for further elucidating the mechanism of steroid induced diabetes mellitus. Methods (1) Rat islet 尾 cell line INS-1 cells were selected as the study object. The proliferation of INS-1 cells at different concentrations (0. 01 渭 M) DEX) (0. 01 ~ 0. 1 渭 M) DEX) for 3 days was detected, and the optimal concentration and time of DEX were determined. (2) the death of INS-1 cells was detected by trypan blue staining, Tunel staining and flow cytometry after treatment with different concentrations of DEX for 48 h. (3) the expression of GSK-3 尾 and p-GSK-3 尾 (Ser9) in INS-1 cells induced by DEX was observed by immunofluorescence staining. The expression of SOD,i NOS,Nox4,NADPH oxidase (p47phox) and GSK-3 尾 m RNA in INS-1 cells were detected by Real-time PCR method after treatment with 40.1 渭 M DEX for 48 h. The expression of GSK-3 尾, p-GSK-3 尾 (Ser9), SOD,i NOS and Nox4 were detected by western-blot method, and the release levels of ROS and NO were detected by ROS kit and Griess assay. 5 the changes of the above indexes were observed after adding GSK-3 尾 inhibitor Li Cl. Results the survival rate of INS-1 cells decreased with the increase of DEX dose and time after treatment with DEX (0. 01 ~ 0. 1 渭 M) for 1 to 3 days. Tunel staining and flow cytometry showed that the apoptosis of INS-1 cells was observed at the concentration of 0.1 渭 m DEX for 48 h. Compared with the control group, the level of ROS,NO in the supernatant of cell culture was increased, and the level of Nox4,p47phox, in the supernatant was higher than that in the control group. The expression of i NOS m RNA was up-regulated and the expression of, SOD m RNA was down-regulated. I NOS,Nox4 protein expression increased, SOD,p-GSK-3 尾 (Ser9) protein expression decreased significantly, total GSK-3 尾 protein expression did not change significantly. After the addition of Li Cl, the apoptosis rate induced by DEX was significantly decreased, the level of ROS,NO was decreased, the expression of Nox4,p47phox,i NOS m RNA and I NOS,Nox4 protein were down-regulated, and p-GSK-3 尾 (Ser9) protein expression was up-regulated. The difference was statistically significant (P0.05). Conclusion Dexamethasone can induce apoptosis of islet 尾 cells in rats. The mechanism may be related to the increase of oxidative stress and the activity of GSK-3 尾. Inhibiting the activity of GSK-3 尾 can reduce the apoptosis induced by dexamethasone to some extent, and its mechanism may be related to the reduction of oxidative stress.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
【参考文献】
相关期刊论文 前1条
1 杨红旗;臧卫周;徐军;;雌激素对H_2O_2诱导的氧化应激的保护作用及机制[J];神经解剖学杂志;2008年05期
，本文编号：2429289
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2429289.html
最近更新
教材专著
