胰岛素对谷氨酸诱导PC12细胞损伤的保护作用及其机制

背景目前越来越多的研究表明谷氨酸可以诱导细胞凋亡,对胰岛素的间接和直接的神经保护作用也有较多的研究,然而对谷氨酸损伤模型的胰岛素保护作用及机制尚缺乏有意义的探讨.目的明确胰岛素对于谷氨酸诱导PC12细胞损伤模型有无保护作用及初步探讨保护作用的分子机制.设计以细胞为研究对象,前瞻性对照实验研究.单位浙江医院神经科和中山大学医院神经科.材料实验于2002-03/2003-03在中山附属第三医院实验室及中山大学实验动物中心完成.PC12细胞来自中山大学医学院实验动物中心.方法用0.5mmol/L谷氨酸作用PC12细胞20 min制成谷氨酸损伤模型,用不同浓度胰岛素保护,24h后分别进行MTT实验、Hoechst33258荧光染色、DNA琼脂糖凝胶电泳及检测PKB/Akt蛋白表达.主要观察指标①各组细胞的活力.②DNA的提取和琼脂糖凝胶电泳结果.③各实验组PKB/Akt蛋白表达结果.结果胰岛素50mU/L,100 mU/L, 200 mU/L, 400 mU/L的A值分别是0.214±0.062,0.234±0.067,0.260±0.076,0.265±0.069,而单纯谷氨酸组的A值为0.201±0.079,进行统计学分析表明50mU/L,100 mU/L胰岛素组与谷氨酸组比较无差异(P>0.05),200 mU/L,400 mU/L胰岛素组与单纯谷氨酸组比较统计学有差异(t=-2.398,-2.716,P<0.05);400mU/L胰岛素组DNA电泳中未见"DNA Ladder";发现胰岛素可以促进PKB/Akt蛋白表达.结论胰岛素对谷氨酸诱导的PC12细胞损伤模型有保护作用,且胰岛素有抗凋亡作用,此作用可能与胰岛素促进PKB/Akt蛋白表达有关.

Protective effects and mechanism of insulin on impairment induced by glutamine in PC12 cells

BACKGROUND: More and more researches prove that cell apoptosis could be induced by glutamine, also there are more researches on studying the indirect and direct nervous-protective effects of insulin, but the nervous-protective effects of insulin on impairment induced by glutamine, as well as its mechanism still need further investigation.OBJECTIVE: To investigate the nervous-protective effects of insulin on impairment induced by glutamine in PC12 cells, and to explore its molecular mechanism.DESIGN: A prospective controlled study based on cells.SETTING: Department of Neurology, Zhejiang Hospital; Department of Neurology of Sun Yat-wen University Hospital.MATERIALS: The study was carried out at the Laboratory of the Third Affiliated Hospital and the Experimental Animal Center of Sun Yat-sen University from March 2002 to March 2003. PC12 cells were purchased from the same animal center.METHODS: Traumatic models were made in PC 12 cells by treated with 0.5 mmol/L glutamine for 20 minutes, and the insulin of different concentration were used for protection, after 24 hours, protective effects of insulin were assessed with MTT method, Hoechst33258 fluorescence staining, DNA agar gelatin electrophoresis, meanwhile the expression of PKB/Akt protein were also detected./Akt protein in experimental group.RESULTS: The A value of50 mU/L, 100 mU/L, 200 mU/L, 400 mU/L insulin groups were 0. 214 ±0. 062, 0. 234 ±0. 067, 0. 260 ±0. 076 and 0. 265 ± 0. 069, respectively, but the value of single glutamine group was 0. 201 ± 0. 079, statistical analysis indicated that compared with single glutamine group, there were no significant difference in 50 mU/L, 100 mU/L insulin groups( P > 0.05), but 200 mU/L, 400 mU/L insulin groups were found statistically different from single glutamine group(t=-2.398,-2. 716, P < 0.05); "DNA Ladder" could not be observed in 400 mU/L insulin group by electrophoresis;It was proved that Insulin could enhance the expression of PKB/Akt protein.CONCLUSION: Insulin has nervous-protective effects on impairment induced by glutamine in PC12 cells, furthermore it also has property of anti-apoptosis, and its protective mechanism might be associated with enhancement of the expression of PKB/Akt protein.