【摘要】：目的應用地塞米松(DEX)誘導的大鼠胰島INS-1細胞凋亡模型。探討糖原合酶激酶-3β(GSK-3β)在糖皮質激素誘導胰島β細胞凋亡過程中的作用及可能的分子機制,為進一步闡明類固醇性糖尿病的發(fā)生機制提供實驗依據(jù)。方法1選用大鼠胰島β細胞株INS-1細胞作為研究對象,首先應用MTT法檢測不同濃度(0、0.01、0.1、1μM)DEX作用細胞1,2,3d時細胞增殖情況,確定DEX的最佳作用濃度和作用時間;2不同濃度DEX作用INS-1細胞48h后通過臺盼藍染色、Tunel染色和流式細胞術檢測細胞死亡情況;3免疫熒光染色方法觀察DEX誘導后,INS-1細胞中GSK-3β和p-GSK-3β(Ser9)的蛋白表達;4 0.1μM DEX處理INS-1細胞48h后,采用Real-time PCR方法檢測SOD、i NOS、Nox4、NADPH oxidase(p47phox)和GSK-3βm RNA的表達;采用western-blot方法檢測GSK-3β、p-GSK-3β(Ser9)、SOD、i NOS和Nox4蛋白的表達;采用ROS試劑盒、Griess法檢測ROS及NO的釋放水平。5觀察加入GSK-3β抑制劑Li Cl后上述各項指標變化。結果DEX(0、0.01、0.1、1μM)作用INS-1細胞1--3天,隨著DEX劑量和時間增加,INS-1細胞存活率下降;Tunel染色、流式細胞術證實0.1μΜ濃度的DEX作用INS-1細胞48h時,細胞出現(xiàn)明顯的凋亡現(xiàn)象,并且與對照組相比,細胞培養(yǎng)上清中的ROS、NO水平升高,Nox4、p47phox、i NOS m RNA表達上調,SOD m RNA表達下調;i NOS、Nox4蛋白表達水平升高,SOD、p-GSK-3β(Ser9)蛋白表達明顯降低,總GSK-3β蛋白表達無明顯變化。加入Li Cl后DEX誘導的細胞凋亡率明顯下降,ROS、NO水平降低,Nox4、p47phox、i NOS m RNA表達及i NOS、Nox4蛋白表達均明顯下調,p-GSK-3β(Ser9)蛋白表達上調,差異均具有統(tǒng)計學意義(P0.05)。結論地塞米松可誘導大鼠胰島β細胞凋亡,其機制可能與氧化應激反應增強、GSK-3β活性增加有關;抑制GSK-3β活性在一定程度上可減輕地塞米松誘導的細胞凋亡,其機制可能與減輕氧化應激反應有關。
[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.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1

【參考文獻】

相關期刊論文 前1條

1 楊紅旗;臧衛(wèi)周;徐軍;;雌激素對H_2O_2誘導的氧化應激的保護作用及機制[J];神經(jīng)解剖學雜志;2008年05期

，

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