缺氧早期心肌细胞微管结构变化对糖酵解的影响及其机制的研究

发布时间：2018-01-25 08:35

本文关键词： 心肌细胞缺氧微管糖酵解 HIF-1α 能量代谢微管干预剂　出处：《第三军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 目的缺氧是许多疾病过程中重要的病理生理现象之一,缺氧诱导因子(HIF)-1α在缺氧细胞的能量代谢中有着重要作用。本研究旨在明确缺氧早期心肌细胞微管结构破坏是否通过调控HIF-1α来影响细胞糖酵解供能。材料和方法 1、建立体外培养新生大鼠心肌细胞模型。分别采用常氧、缺氧、以及常氧和缺氧下微管解聚剂和不同浓度微管稳定剂处理心肌细胞;建立心肌细胞高表达微管相关蛋白4和RNA干扰后低表达α-微管蛋白模型。 2、激光扫描共聚焦显微镜观察培养新生大鼠心肌细胞α-微管结构和含量变化,胎盘蓝染色观察细胞存活率,CCK法观察细胞活力,化学比色法和高效液相色谱法分别观察心肌细胞糖酵解关键酶(PK、HK和PFK)活性、肌酸激酶(CK)、乳酸生成,LDH漏出及ATP/ADP生成。 3、免疫印记法及激光共聚焦显微镜观察心肌细胞微管结构改变后HIF-1α蛋白含量和细胞内分布变化,实时定量PCR法检测心肌细胞HIF-1αmRNA表达变化。结果 1、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏,聚合态α-微管蛋白含量减少,心肌细胞的活力降低,死亡率升高;稳定微管网状结构可以使细胞存活率和活性升高。 2、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏引起糖酵解关键酶(PK、HK和PFK)活性降低,代谢终产物乳酸生成减少,细胞ATP生成减少;而稳定微管网状结构可以在缺氧早期一段时间内升高PK、HK和PFK的活性,促进细胞能量生成。 3、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏使HIF-1α蛋白表达及入核表达均减少。微管稳定剂和高表达微管相关蛋白4可稳定缺氧心肌细胞微管网状结构,上调HIF-1α蛋白含量及入核表达;微管解聚剂和下调微管蛋白表达则可加重缺氧心肌细胞微管结构破坏,HIF-1α蛋白含量及入核表达减少更明显。而且HIF-1α蛋白表达的增加发生在转录后水平。结论微管结构变化通过调节HIF-1α可影响缺氧心肌细胞早期糖酵解。稳定微管结构可促进HIF-1α入核表达并提高HIF-1α蛋白含量,提高厌氧糖酵解关键酶活性和能量生成,表明微管结构变化通过调节HIF-1α影响缺氧心肌细胞早期糖酵解,这为临床改善缺氧早期细胞能量代谢提供了潜在的治疗靶点。
[Abstract]:Purpose Hypoxia is one of the important pathophysiological phenomena in many diseases. Hypoxia inducible factor (HIFs). 1 伪 plays an important role in the energy metabolism of anoxic cells. The purpose of this study was to determine whether the destruction of microtubule structure of cardiomyocytes in the early stage of hypoxia affects the glycolytic energy supply by regulating HIF-1 伪. Materials and methods 1. The neonatal rat cardiomyocyte model was established in vitro. Cardiomyocytes were treated with normoxic, hypoxia, microtubule depolymerization agent and microtubule stabilizer of different concentrations. The model of low expression of 伪-tubulin in cardiomyocytes with high expression of microtubule-associated protein 4 and RNA interference was established. 2. The changes of 伪 -microtubule structure and content in cultured neonatal rat cardiomyocytes were observed by laser scanning confocal microscopy and the viability of cultured neonatal rat cardiomyocytes was observed by CCK method. The activities of PKHK and PFK, creatine kinase (CK) and lactic acid (lactic acid) were measured by chemical colorimetry and high performance liquid chromatography (HPLC), respectively. LDH leakage and ATP/ADP formation. 3Immunoimprinting and laser confocal microscopy were used to observe the changes of HIF-1 伪 protein content and intracellular distribution after microtubule structure changes in cardiomyocytes. The expression of HIF-1 伪 mRNA in cardiomyocytes was detected by real time quantitative PCR. Results 1. At the early stage of hypoxia, the microtubule reticular structure of neonatal rat cardiomyocytes in vitro was destroyed, the content of polymeric 伪 -tubulin was decreased, the activity of cardiomyocytes was decreased, and the death rate was increased. Stable microtubule reticular structure can increase cell viability and activity. 2, early after hypoxia, the destruction of microtubule reticular structure of neonatal rat cardiomyocytes in vitro resulted in the decrease of PKK and PFK activities, and the decrease of lactic acid production of the end product of metabolism. Cell ATP production decreased; The stable microtubule reticular structure could increase the activity of PKHK and PFK in the early stage of hypoxia and promote cell energy production. 3, early after hypoxia. The destruction of microtubule reticular structure in neonatal rat cardiomyocytes in vitro reduced the expression of HIF-1 伪 protein and the expression of microtubule-associated protein 4. Microtubule-associated protein 4 and microtubule stabilizer could stabilize the microtubule reticular node in hypoxic cardiomyocytes. Structure. Upregulation of HIF-1 伪 protein content and expression in nucleus; Microtubule depolymerization agent and down-regulation of tubulin expression can aggravate the destruction of microtubule structure in hypoxic cardiomyocytes. The decrease of HIF-1 伪 protein and the expression of HIF-1 伪 protein were more obvious, and the increase of HIF-1 伪 protein expression occurred at posttranscriptional level. Conclusion The change of microtubule structure can affect the early glycolysis of hypoxic cardiomyocytes by regulating HIF-1 伪. Stabilizing the microtubule structure can promote the expression of HIF-1 伪 and increase the protein content of HIF-1 伪. The key enzyme activity and energy production of anaerobic glycolysis were increased, which indicated that the change of microtubule structure affected the early glycolysis of hypoxic cardiomyocytes by regulating HIF-1 伪. This provides a potential therapeutic target for clinical improvement of early anoxic cell energy metabolism.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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