摘要
目的：通过第三丁基过氧化氢（t-BHP）诱导小鼠胰岛β细胞凋亡,研究氧化损伤对内质网应激JNK凋亡通路相关分子表达的影响。方法：将不同浓度t-BHP（0-400μmol/L）分别加入胰岛β细胞株M IN6细胞中,于不同时间（0-8 h）收集细胞悬液进行相关检测。CCK-8法检测细胞增殖活力;An-nexin-V-PI流式细胞技术（FCM）检测细胞凋亡率及坏死率;Caspase-3检测试剂盒测定caspase-3活性;W estern b lot检测内质网应激相关分子IRE1,αJNK,P-JNK,Caspase-3蛋白的表达。结果：随t-BHP浓度的增高,①M IN6细胞的存活率降低;②t-BHP以浓度≥25μmol/L作用≥1 h时,Caspase-3活性有明显变化（P〈0.05）;③随t-BHP浓度增大、作用时间延长,内质网应激跨膜蛋白IRE1α表达逐渐减少,P-JNK、活性caspase-3表达明显增多。结论：①t-BHP引发细胞凋亡坏死呈一定的时效和量效关系;②持续t-BHP氧化损伤可诱导M IN6细胞发生内质网应激并发生凋亡;③内质网应激凋亡通路相关分子表达在细胞凋亡过程中有浓度和时间依赖性。
AIM： Through a third-butyl hydrogen peroxide（t-BHP） induced apoptosis in pancreatic islet β-cells to study the oxidative damage induced endoplasmic reticulum stress-JNK pathway of apoptosis related molecules in vitro.METHODS： Mouse insulinoma（MIN6） cells was administered with t-BHP which were cultured in vitro.Choosing medicine with different concentrations（0-400 μmol/L）and time periods（0-8 h）to establish the cells apoptosis model.The percentage of cell viability was determined through CCK-8 assay.The percentage of apoptosis was determined through flow cytometric assay after Annexin-Ⅴ-FITC-PI staining.The activity of caspase-3 was measured by the caspase-3 activity assay kit.The expression of Endoplasmic reticulum stress-related molecules and the apoptosis signal pathway IRE1,JNK,P-JNK,Caspase-3 were detected by Western blot.RESULTS： The percentage of MIN6 cell viability was reducing with the concentration of t-BHP increasing.The Caspase-3 significantly change the activity after exposured of t-BHP in a concentration ≥25 μmol/L when the role of ≥1 h,With t-BHP concentration was increased,the role of prolonged,endoplasmic reticulum stress transmembrane protein IRE1α,P-JNK,active caspase-3 expression was significantly increased.CONCLUSION： The study demonstrates that the percentage of MIN6 cell viability was reduced in a dose-dependent manner.Continuous exposuring of t-BHP induced oxidative damage in MIN6 cells to endoplasmic reticulum stress and apoptosis.The expression of Endoplasmic reticulum stress and apoptosis pathway-related molecules in cell apoptosis in a dose and time-dependent.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第3期249-252,256,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
福建省高校新世纪优秀人才资助项目(NCETFJ0703)
