论文部分内容阅读
目的 探讨αB 晶状体蛋白抗心肌细胞凋亡的分子机制。方法 构建带 6个串联组氨酸的αB 晶状体蛋白表达质粒 (pcDNA3.1 aBC) ,经测序证实后 ,转染C2 C12 肌原细胞株 ,筛选稳定高表达αB 晶状体蛋白的细胞株。采用H2 O2 (0 .5mmol/L)诱导细胞凋亡 ,检测线粒体细胞色素C释放率和细胞膜磷脂酰丝氨酸细胞色素荧光强度作为细胞凋亡早期判断指标。提取细胞线粒体蛋白及胞浆蛋白 ,采用镍金属亲和层析分离αB 晶状体蛋白与其相互作用蛋白复合物 ,免疫共沉淀证实其蛋白质相互作用。结果 ①H2 O2 处理 1h后 ,转染αB 晶状体蛋白的细胞线粒体释放细胞色素C和细胞膜磷脂酰丝氨酸外翻均较对照组明显减少。②经Westernblot检测 ,发现αB 晶状体蛋白与其相互作用蛋白复合物中存在线粒体凋亡相关蛋白p5 3、核因子κB、细胞色素C和Smac。③经免疫共沉淀证实 ,H2 O2 诱导细胞凋亡时 ,αB 晶状体蛋白与p5 3、细胞色素C和Smac等促凋亡蛋白相互结合增多 ,而与抗凋亡蛋白核因子κB相互结合减少。结论 αB 晶状体蛋白基因转染可早期抑制氧化应激所致的C2 C12 肌原细胞凋亡 ,其机制可能涉及αB 晶状体蛋白与上述凋亡相关蛋白之间的相互作用。
Objective To investigate the molecular mechanism of αB crystallin in cardiomyocyte apoptosis. Methods αB crystallin expression plasmid (pcDNA3.1 aBC) with 6 tandem histidines was constructed. After confirmed by sequencing, the C2C12 myoblast cell line was transfected, and the cell line stably expressing αB crystallin was screened. H2O2 (0. 5mmol / L) was used to induce cell apoptosis, and the mitochondrial cytochrome C release rate and the cell membrane phosphatidylserine cytochrome fluorescence intensity were measured as early indicators of cell apoptosis. The mitochondrial proteins and cytoplasmic proteins were extracted, and the α B crystallin and its interacting protein complexes were separated by nickel metal affinity chromatography, and co-immunoprecipitation was used to confirm the protein interactions. Results ① After treated with H2O2 for 1h, the mitochondria release of cytochrome C and the phosphatidylserine ectopy of the cells transfected with αB crystallin were significantly decreased compared with the control group. ②Western blot analysis showed that the mitochondrial apoptosis-related proteins p5 3, NF-κB, cytochrome C and Smac were present in αB crystallin and its interacting protein complexes. ③ Co-immunoprecipitation confirmed that α2-crystallin interacted with the pro-apoptotic proteins such as p5 3, cytochrome C and Smac, and decreased the binding of anti-apoptotic protein NF-κB to H2O2-induced apoptosis. Conclusion The αB crystallin gene transfection can early inhibit the apoptosis of C2C12 myogenic cells induced by oxidative stress, and its mechanism may involve the interaction between αB crystallin and the above apoptosis related proteins.