缺氧过程中大鼠脑皮质线粒体细胞色素氧化酶活性及其亚基表达协调性的研究

【作者】 谭小玲；

【导师】 柳君泽；

【作者基本信息】 第三军医大学 ， 病理生理学， 2001， 硕士

【摘要】 缺氧可致中枢神经系统功能障碍，而能量生成不足是其主要原因。线粒体是能量产生的主要场所，缺氧时脑线粒体氧化磷酸化功能改变的确切机制尚不清楚。线粒体结构和功能的完整相当程度上依赖于呼吸链上酶功能的正常发挥。由mtDNA和nDNA编码的13个蛋白亚基组成的细胞色素氧化酶（COX）是呼吸链上的关键酶，其亚基的正确匹配对酶功能的正常发挥极其重要，也是线粒体能量合成的基础。我们通过观察缺氧暴露不同时间大鼠脑组织COX活性、COX亚基Ⅰ和Ⅳ的基因表达状况，藉以了解缺氧过程中COX活性与其基因表达的关系、缺氧对mtDNA和nDNA编码COX亚基表达协调性的变化，以阐明缺氧时COX在线粒体氧化呼吸功能改变中的作用。11-14周龄的成熟Wistar大鼠50只随机分为平原组、缺氧2天组、缺氧5天组、缺氧15天组和缺氧30天组。缺氧组于模拟5000米高原低压舱（大气压405.35mmHg）内连续缺氧24小时/天，于舱内取材，平原组动物于舱外喂养及取材。采用本室建立的方法提取脑皮质组织线粒体；极谱法测量COX活性；Wesrern Blot分析线粒体内COXⅠ和COXⅣ蛋白量；RT-PCR检测COXⅠ和COXⅣmRNA稳态量（statelevel）。主要结果：1、大鼠经缺氧2天、5天、15天时，脑组织COX活性降低，与平原对照组相比，差异非常显著，缺氧15天时达到最低，只有平原对照组的58％；缺氧30天时，COX活性与缺氧15天组相比活性升高，但仍低于平原对照组，只有平原对照组的79％。二、缺氧 2天和 5天时，COX mRNA稳态量与平原对照组相比显著增加。 缺氧 15天和 30天时 mRNA稳态量恢复到平原对照水平；随缺氧时间 的延长，COXwmRNA稳态量增加，在缺氧15天时达到高峰，缺氧30 天时下降并低于平原对照水平；COXIV／COX mRNA量比值在缺氧 15 天时显著升高，其它缺氧组与平原组及其组间差异无显著意义；12sr RNA在缺氧2天时增加，与平原对照组差异有显著意义，其它各组与 平原对照组差异无显著意义。3、缺氧过程中，线粒体内 COX和 COXIV蛋白含量没有显著变化。COX IVCOX蛋白比值在各组间差异无显著意义。结 论1、缺氧时大鼠脑皮质线粒体COX活性降低，并且与缺氧存在时间依赖 关系，在缺氧 15天以内呈进行性降低，缺氧 30天时 COX活性有部分 恢复，表明缺氧过程中COX活性改变是线粒体氧化呼吸和能量生成改 变的原因之一。二、缺氧并不引起大鼠脑皮质线粒体内 COX、IV亚基蛋白含量的改变， 提示COX活性的变化主要是通过定性调节的。3、缺氧早期门天、5天L COX、IV mRNA稳态量呈相协调性升高， 而缺氧 15天时发生失调，但缺氧过程中蛋白水平上 COX！、IV及其 比值的相对恒定，则提示COX亚基表达及其协调性的调节存在转录后 或蛋白水平上的调整。更多还原

【Abstract】 To understand the mechanism of disorder in brain energy metabolism and explore the contributing role of COX in disorder in mitochondrial respiratory function during hypoxic exposure. We observed the brain cortex mitochondrial COX activity, COX subunits gene expression ecoded by mtDNA and nDNA during rat exposed to hypoxia. Adult male Wistar rats were exposed to simulated high altitude at 5000m for 0(control), 2(2d), 5(5d), 15(1 Sd) and 30 days(30d) in hypobaric chamber. Animals were sacrificed by decaptation under normoxic(control) and hypoxic(other groups)conditions respectively Rats brain cortex was removed and mitochondria were isolated by centrifugation. COX activity was measured by the Clark oxygen electrode. The protein content of COX subunit I and IV in mitochondria were detected by Western blot analysis and mRNA state level of the two COX subunits( I and IV) in tissues by RT.-PCR. The results showed that: 1) Within the 15 days hypoxic exposure, COX activity decreased significantly than control, expecially in I Sd group animals; but restored to 78% of the control level in 30d group animals; 2) During hypoxic exposure , the protein content of COX subunit I and IV in mitochondria from brain cortex did not change ; the ratio of subunit IV/ I also had no significant differences among each groups; 3)The subunit I mRNA state level increased significantly in 2d and Sd than in control, but decreased to control level in 15d and 30d group ; Subunit IV mRNA state level of animals in 2d, Sd and 1 Sd group were dramatically higher than that in control, but lower in 30d group; l2sr RNA state level was higher in 2d group than in control, Sd, 15.d and 30d, but no sinificant differences among control, Sd, 15d and 30d group. Except higher in 1 Sd group than in control, the ratio of mRNA state level of COX subunit IV to I had no significant differences between other groups. Conclusion: 1) COX activity in rat brain cortex decreased during rat exposed to hypoxia .Within 15 days hypoxic exposure, COX activity decreased progressly and then partly recovered when rat further exposure. It showed that the changes of COX activity in rat brain cortex were time-dependent with hypoxic exposure. This results indicated that the changes contribute to the disorder of mitochondrial respiratory function and energy pruduction during hypoxia; 2) The content of COX subunit I and IV protein in mitochondria from rat brain cortex had no differences among each groups of hypoxic exposed animals , which suggested that qualitative control in regulation of COX activity might be the major model during hypoxic exposure; 3) In early of hypoxic exposure (2 days and Sdays), COX subunit I and IV mRNA state level increased coordinately, but disproportionly at 15 days hypoxic exposure. While the content of COX subunit I and IV protein and their ratio in mitochondria from rat brain cortex during hypoxic exposure had no change. This concluded that the regulation of COX subunits gene expression and the their coordination might be adjusted at post-transcription level or protein level.更多还原