机构地区:[1]华中科技大学同济医学院附属同济医院呼吸内科,武汉430030
出 处:《中华结核和呼吸杂志》2006年第11期727-730,共4页Chinese Journal of Tuberculosis and Respiratory Diseases
基 金:国家自然科学基金资助项目(30370623)
摘 要:目的研究线粒体膜上ATP敏感钾通道(MitoKATP)的开放剂二氮嗪和线粒体膜电位在缺氧引起的人肺动脉平滑肌细胞(HPASMC)内氧自由基的变化及细胞增殖/凋亡失衡中的作用。方法培养HPASMC并将所培养的细胞分为6组正常对照组(A组);MitoKATP阻断剂5-羟基癸酸盐(5-HD)组(B组);MitoKATP开放剂二氮嗪组(C组);慢性缺氧组(D组);慢性缺氧+二氮嗪组(E组);慢性缺氧+5-HD组(F组),每组样本数均为6。利用激光共焦显微镜成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,免疫组化法检测增殖细胞核抗原(PCNA)、c-fos及c-jun的蛋白表达和四甲基偶氮唑盐(MTT)法检测细胞增殖情况。结果C、D、E组细胞线粒体膜电位(以R123的荧光强度表示)分别为105±4、95±13、126±8,较A组(75±7)明显去极化(q值分别为5.474、3.659、9.213,P均<0.05);C、D、E组细胞内氧自由基含量分别为3045±126、3116±34、3236±31,与A组(2772±49)比较差异有统计学意义(q值分别为6.882、7.448、16.289,P均<0.05);C、D、E组细胞增殖活性[以MTT法检测出的A值表示]分别为0.305±0.022、0.328±0.078、0.440±0.023,与A组(0.237±0.013)比较差异有统计学意义(q值分别为2.993、4.017、8.919,P均<0.05),且E组线粒体膜电位、细胞内氧自由基含量、细胞增殖活性与D组比较差异有统计学意义(q值分别为5.554、8.841、4.902,P均<0.05)。F组线粒体膜电位、细胞内氧自由基含量、细胞增殖活性分别为71±4、2863±132、0.264±0.045,与D组(95±13、3116±34、0.328±0.078)比较差异有统计学意义(q值分别为4.367、5.907、2.832,P均<0.05)。结论二氮嗪或缺氧能够通过开放HPASMC线粒体膜上ATP敏感的钾通道,引起线粒体膜电位去极化,增加细胞内氧自由基的含量,最终导致HPASMC的增殖/凋亡的失衡,从而促进了缺氧性肺动脉重塑过程。Objective To investigate the contribution of the opening of mitochondrial ATP-sensitive K ^+ channel( MitoKATP ) and mitochondrial membrane potential(△ψm) to changes of reactive oxygen species (ROS) and to the imbalance of proliferation/apoptosis of human pulmonary arterial smooth muscle cells (HPASMC) induced by hypoxia. Methods HPASMC were divided into the following groups : ( 1 ) control group ( A group ) : cultured under normoxia; ( 2 ) 5-HD group ( B group ) : cultured in normoxia with 5-hydroxydecanoate ( 5 -HD ), an antagonist of MitoKATP, for 24 h ; ( 3 ) diazoxide group ( C group) : cultured in normoxia with diazoxide, an opener of MitoKATp ,for 24 h; (4) chronic hypoxia group ( D group) :cultured under hypoxia for 24 h; ( 5 ) chronic hypoxia + diazoxide group( E group) : cultured in hypoxia with diazoxide for 24 h;( 6 )chronic hypoxia + 5-HD group( F group) : cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence(R-123) technique. The level of ROS in HPASMC was tested with chemiluminescence method. The proliferation of HPASMC was examined by the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay. Results The mitochondrial membrane potentials(expressed by intensity of R-123) of C group ( 105 ±4) , D group (95 ±13) and E group (126 ±8) were significantly depolarized as compared with A group (75 ±7, q =5. 474, 3.659, 9.213, all P〈0.05). The levels ofROS of C group (3 045 ±126) ,D group (3 116 ±34) and E group (3 236 ± 31 ) were significant increased than that of A group (2 772 ± 49 ) (q = 6. 882, 7.448, 16. 289,all P 〈0.05) . The cell viability of C group (0. 305 ±0. 022), D group (0. 328 ±0. 078) and E group (0. 440 ±0. 023) were significant increased than that of A group (0. 237 ±0. 013) in HPASMC (q = 2. 993, 4. 017,8.919, all P 〈 0. 05 �
分 类 号:R543.2[医药卫生—心血管疾病]
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