【作者】 刘亮；

【导师】 刘礼斌；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:探讨PI3K/Akt/Bcl-2信号通路在游离脂肪酸诱导的小鼠胰岛β细胞株MIN6细胞凋亡中的作用;在此基础上,进一步研究GLP-1受体激动剂Exendin-4对FFA诱导的MIN6细胞凋亡的作用及其可能的机制。方法:选用小鼠胰岛β细胞株MIN6细胞为研究对象,用不同浓度(0.2-1.6mmol/L)FFA诱导刺激,模拟体外β细胞脂毒性损伤后的细胞凋亡模型,通过MTT观察FFA作用后细胞的存活率;Hoechst-PI染色荧光显微镜观测细胞凋亡形态,Annexin-Ⅴ-PI染色流式细胞技术测定细胞凋亡率,Western blot技术检测胞浆Akt、P-Akt、Bcl-2和Bax蛋白表达水平的变化。在此基础上,用不同浓度(12.5-200nmol/L)的Exendin-4预处理细胞,观察Exendin-4对FFA诱导的MIN6细胞凋亡的保护作用,并选定一个合适的浓度进一步研究Exendin-4是否通过PI3K/Akt/Bcl-2信号通路影响FFA诱导MIN6细胞发生凋亡性损伤。结果:MTT显示FFA(0.4-1.6mmol/L)作用不同时间后可明显抑制MIN6细胞的生长,并呈现一定的量效关系;Hoechst-PI染色荧光显微镜及Annexin-Ⅴ-PI双染流式细胞检测证实不同浓度的FFA处理48小时后,随着FFA浓度的增高,其诱导MIN6细胞的凋亡作用就更强。不同浓度的FFA处理48小时后,随着FFA浓度的增加,细胞内P-Akt、Bcl-2蛋白水平降低,而Akt、Bax蛋白水平变化不明显。不同浓度的Exendin-4预处理24小时,可以抑制FFA诱导的β细胞凋亡,并呈现一定的剂量依赖性,并且Exendin-4能抑制FFA诱导的胞内P-Akt、Bcl-2蛋白水平的降低。结论:(1)FFA可诱导胰岛β细胞发生凋亡性损伤,在此过程中伴随细胞内P-Akt、Bcl-2蛋白水平的降低;(2)PI3K/Akt/Bcl-2信号通路可能是FFA诱导胰岛β细胞发生凋亡的重要通路之一;(3)GLP-1受体激动剂Exendin-4可以剂量依赖性地抑制FFA诱导胰岛β细胞的凋亡;(4)Exendin-4可能通过激活细胞内Akt的活化、提高胞浆Bcl-2蛋白表达水平来抑制FFA诱导的β细胞凋亡。Exendin-4在胰腺β细胞保护治疗中具有潜在的应用价值。更多还原

【Abstract】 OBJECTIVE: To investigate the effects of free fatty acid(FFA)on cell apoptosis in murine MIN6 pancreaticβ-cells and the changes in signaling pathway of Akt /Bcl-2 in this process.Further, to explore the effect and possible mechanism of Exendin-4 onβ-cells apoptosis induced by FFA.METHODS: Murine MIN6 pancreaticβ-cells were cultured in vitro and the cell apoptosis model of lipotoxicity was established by FFA. Cells were pretreated with Exendin-4 at the concentration of 12.5 to 200nmol/L.Choose a correct concentration of Exendin-4 to continue the following study.Cell viability was measured by MTT assay.The morphological changes of cell damage was evaluated by epifluorescence microscopy after staining with Hoechst-PI.The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. Protein levels of Akt、P-Akt、Bcl-2 and Bax were detected by Western blot.RESULTS: MIN6 cells viability were significantly reduced in a dose-dependent manner after exposure of MIN6 cells to free fatty acid at range of 0.4-1.6 mmol/L for 24 and 48 hours.It was verified by Hoechst-PI and Annexin-Ⅴ-FITC-PI staining that the percentage of cell apoptosis was significantly increased after treating with FFA. Meanwhile,the protein levels of P-Akt、Bcl-2 but Akt、Bax were dose-dependently decreased.Intriguingly, cell apoptosis was dose-dependently reduced by being pretreated 24 hours with Exendin-4.Furthermore,the present study showed that Exendin-4 patently inhibited the decreased protein levels of P-Akt、Bcl-2 induced by FFA.CONCLUSION: The cell signaling pathway of Akt/ Bcl-2 mediated FFA-induced apoptosis in MIN6 pancreaticβ-cells. Exendin-4 protectedβ-cells from FFA-induced apoptosis via up-regulation of Akt/ Bcl-2 pathway.Glucagon-like peptide-1 receptor agonist—Exendin-4 is potentially a highly effective therapeutic agent in protecting pancreaticβ-cells.更多还原