论文部分内容阅读
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 effects of diazoxide and mitochondrial membrane potential on mitochondrial ATP sensitive potassium channel (MitoKATP) openers on oxygen free radicals and cell proliferation / apoptosis imbalance induced by hypoxia in human pulmonary artery smooth muscle cells (HPASMC) . Methods HPASMCs were cultured and divided into six groups: normal control group (group A), MitoKATP blocker 5-hydroxydecanoate (group B), MitoKATP opener diazoxide group (Group C); chronic hypoxia group (group D); chronic hypoxia + diazoxide group (group E); chronic hypoxia + 5-HD group (group F) Mitochondrial membrane potential was detected by laser scanning confocal microscopy. The content of oxygen free radicals in cells was detected by fluorescence staining. The protein expressions of proliferating cell nuclear antigen (PCNA), c-fos and c-jun were detected by immunohistochemistry and the protein expression of tetramethylpyrazole Salt (MTT) method to detect cell proliferation. Results The mitochondrial membrane potentials (expressed as fluorescence intensity of R123) in groups C, D and E were 105 ± 4, 95 ± 13 and 126 ± 8, respectively, which were significantly lower than those in group A (75 ± 7) 5.474, 3.659, 9.213, P <0.05). The contents of intracellular oxygen free radicals in groups C, D and E were 3045 ± 126, 3166 ± 34 and 3236 ± 31 respectively, which were statistically different from those in group A (2772 ± 49) (Q = 6.882, 7.448, 16.289 respectively, P <0.05). The proliferative activity of cells in group C, D and E [A value measured by MTT method] was 0.305 ± 0.022,0.328 ± 0.078 and 0.440 ± 0.023, there was significant difference compared with group A (0.237 ± 0.013) (q = 2.993,4.017,8.919, P <0.05), and the mitochondrial membrane potential, content of intracellular oxygen free radicals and cell proliferation The difference between the activity and the D group was statistically significant (q values were 5.554,8.841,4.902, P <0.05). The mitochondrial membrane potential, content of intracellular oxygen free radicals and cell proliferative activity in group F were 71 ± 4,2863 ± 132,0.264 ± 0.045, respectively, which were statistically different from those in group D (95 ± 13,3116 ± 34,0.328 ± 0.078) Significance of learning (q values were 4.367,5.907,2.832, P <0.05). Conclusions Diazoxide or hypoxia can promote the deregulation of HPASMC proliferation / apoptosis by opening the ATP-sensitive potassium channels on the mitochondrial membrane of HPASMC, causing depolarization of mitochondrial membrane potential and increasing intracellular oxygen free radicals A hypoxic pulmonary remodeling process.