线粒体途径在棕榈酸诱导INS-1细胞凋亡的机制探讨

发布时间：2018-06-17 11:42

本文选题：线粒体途径 + β细胞　；参考：《华中科技大学》2012年硕士论文

【摘要】：目的：观察不同浓度棕榈酸(palmiticacid,PA)对大鼠胰岛INS-1细胞的脂毒性作用，探讨线粒体途径是否介导PA诱导的胰岛β细胞INS-1凋亡及其可能机制。方法：以大鼠INS-1胰岛β细胞株为实验对象，在含或不含PA(0.25-1.0mmol／l)的RPMI1640无血清培养基体外培养24h，构建游离脂肪酸PA诱导β细胞INS-1脂性凋亡模型；MTT法测定线粒体琥珀酸脱氢酶活力，反映细胞线粒体功能改变；Hoechst33342/PI活细胞荧光染色法观察INS-1细胞核形态改变，测定细胞凋亡率；RT-PCR法检测0.5mM棕榈酸干预下细胞caspase-3、caspase-9、Bcl-2、AIFmRNA的表达。结果：在无血清条件下，棕榈酸干预24h能显著诱导INS-1细胞凋亡。不同浓度PA处理INS-1细胞，镜下可观察到细胞呈浓度依赖的毒性改变。与正常细胞相比，PA组细胞随PA浓度增加形态逐渐消失，多数细胞收缩甚至呈圆形。MTT法测定线粒体琥珀酸脱氢酶活力，不同浓度棕榈酸干预下，OD值降低显著(p0.05)；Hoechst33342染色在荧光显微镜下观察细胞的凋亡细胞核呈致密浓染或呈碎块状致密浓染，可见明显核碎裂。0.5mmol/LPA组细胞的凋亡率可达到56.6%，与同浓度对照BSA组相比有显著差异(p0.01，n=8)；RT-PCR显示促凋亡因子caspase-3、caspase-9、AIF表达较BSA组上调，而抗凋亡因子Bcl-2表达下调，，提示内源性即线粒体凋亡途径参与PA脂毒性效应。结论：PA对INS-1细胞具有明显脂毒性作用，且细胞损伤与凋亡有关，尤其是可能通过线粒体凋亡途径介导胰岛β细胞凋亡。
[Abstract]:Aim: to observe the lipotoxicity of palmitic acididate (PAA) at different concentrations on rat islet INS-1 cells, and to investigate whether the mitochondrial pathway mediates PA-induced apoptosis of islet 尾 -cells and its possible mechanism. Methods: rat INS-1 islet 尾 cell lines were cultured in serum-free medium (RPMI1640) containing or without PAA 0.25-1.0 mmol / L for 24 h in vitro. Free fatty acid PA induced lipid apoptosis of INS-1 尾 cells was established. MTT assay was used to determine the activity of mitochondrial succinate dehydrogenase. The morphological changes of INS-1 cells were observed by Hoechst33342 / Pi fluorescence staining and the expression of caspase-3, caspase-9Bcl-2, AIFmRNA was detected by RT-PCR. Results: the apoptosis of INS-1 cells was induced by palmitic acid for 24 hours without serum. The cytotoxicity of INS-1 cells treated with different concentrations of PA was observed in a concentration-dependent manner. Compared with the normal cells, the morphology of the cells in PA group gradually disappeared with the increase of PA concentration, and most cells contracted or even showed circular .MTT method to measure the activity of succinate dehydrogenase in mitochondria, and the OD value decreased significantly under different concentrations of palmitic acid. Hoechst33342 staining showed that the apoptotic nuclei of BSA cells were densified with dense staining or fragmented dense staining under fluorescence microscope. The apoptotic rate of the cells in the group of nucleus fragmentation. 0.5 mmol / L PA could reach 56.6%, which was significantly different from that of the BSA group at the same concentration. RT-PCR showed that the expression of apoptosis-promoting factor caspase-3, caspase-9 and AIF was up-regulated than that of BSA group, while the expression of anti-apoptotic factor Bcl-2 was down-regulated, suggesting that endogenous mitochondrial apoptotic pathway was involved in PA lipid toxicity. Conclusion it is suggested that the apoptosis of islet 尾 cells may be mediated by the apoptosis of islet 尾 cells mediated by the apoptosis of the islet 尾 cells induced by the apoptosis of the islet 尾 -cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.8

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