氧化应激所致C2C12肌源细胞核仁损伤的分子机制  

The Molecular Mechanism of Nucleolar Impairment Induced by Peroxide Hydrogen in C2C12 Myogenic Cells

作　　者：王慷慨[1] 邓恭华[1] 刘可[1] 易宇欣[1] 鄂顺梅[1] 张玲莉[1] 肖献忠[1] 

机构地区：[1]中南大学湘雅医学院病理生理学教研室,湖南省长沙市410078

出　　处：《中国动脉硬化杂志》2005年第2期133-137,共5页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金 (30 30 0 1 77;30 2 70 533);国家 973重点项目 (G2 0 0 0 0 5690 8);教育部博士点专项基金 (2 0 0 2 0 5330 32 )资助

摘　　要：目的　探讨氧化应激诱导细胞核仁损伤的分子机制。方法　向传代培养的C2C12肌源细胞中加入0 .5mmol/L过氧化氢以模拟氧化应激。结果　甲苯胺兰染色发现正常C2C12细胞可见一个位于中央的,浓染致密的核仁颗粒。过氧化氢处理后3h ,可见明显的核仁分离,处理6h最为明显。细胞总蛋白质合成能力分析发现过氧化氢处理后6h细胞总蛋白质合成能力显著下降(与正常对照比P <0 .0 5 ) ,并持续至2 4h。免疫印迹检测核仁素(又称C2 3)发现,过氧化氢处理1h后可见一80kDa条带,而其110kDa主带明显减弱,过氧化氢处理6h和12h最为明显。用脂质体将核仁素反义核酸导入细胞后2 4h ,免疫印迹发现核仁素表达明显被抑制,同时也可观察到细胞总蛋白合成能力下降及明显的核仁分离。结论　氧化应激通过诱导核仁蛋白质核仁素的裂解并使其110kDa全长分子的量减少,而导致C2C12细胞核仁损伤。Aim To observe the effect of oxidative stress on nucleolar impairment in C2C12 and clarify the possible molecular mechanism. Methods 0.5 mmol/L peroxide hydrogen (H 2O 2) were added into the cultured C2C12 cell lines to mimic oxidative stress. Toluidine Blue staining and total protein synthesis analysis were performed to assess H 2O 2-induced nucleolar structural and functional injury respectively. Immunoblotting and antisense oligonucleotide were used to detect the changes of nucleolar protein nucleolin (also named C23). Results Toluidine Blue staining showed predominantly compact, centrally localized nucleoli in intact control cells, but in H 2O 2-treated cells, an early onset of nucleolar segregation could be found after 3 h and 6 h. Total protein synthesis analysis revealed that compared with normal cells, the capability of cellular protein synthesis is significantly decreased after 6 h treatment with H 2O 2 (P<0.05), and lasted for 24 h. Moreover, an 80 kDa band of nucleolin was found by using immunoblotting after 1 h treatment with H 2O 2, and accompanied by down-regulation of its primary 110 kDa band. The 110 kDa band decreased remarkably after 6 h and 12 h treatment with H 2O 2. After transfected nucleolin antisense oligonucleotides for 24 h and 48 h, expression of nucleolin was down- regulated significantly, and nucleolar segregation and inhibition of total protein synthesis could be observed. Conclusion The molecular mechanism of oxidative stress-mediated nucleolar impairment is related to cleavage and down-regulation of intact nucleolin.

关 键 词：病理学与病理生理学 过氧化氢 核仁 核仁素(C23) C2C12肌源细胞 反义核酸 

分 类 号：R363[医药卫生—病理学]