本文關(guān)鍵詞： 心肌細(xì)胞 缺氧 微管 糖酵解 HIF-1α 能量代謝 微管干預(yù)劑　出處：《第三軍醫(yī)大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】： 目的 缺氧是許多疾病過程中重要的病理生理現(xiàn)象之一,缺氧誘導(dǎo)因子(HIF)-1α在缺氧細(xì)胞的能量代謝中有著重要作用。本研究旨在明確缺氧早期心肌細(xì)胞微管結(jié)構(gòu)破壞是否通過調(diào)控HIF-1α來影響細(xì)胞糖酵解供能。 材料和方法 1、建立體外培養(yǎng)新生大鼠心肌細(xì)胞模型。分別采用常氧、缺氧、以及常氧和缺氧下微管解聚劑和不同濃度微管穩(wěn)定劑處理心肌細(xì)胞;建立心肌細(xì)胞高表達(dá)微管相關(guān)蛋白4和RNA干擾后低表達(dá)α-微管蛋白模型。 2、激光掃描共聚焦顯微鏡觀察培養(yǎng)新生大鼠心肌細(xì)胞α-微管結(jié)構(gòu)和含量變化,胎盤藍(lán)染色觀察細(xì)胞存活率,CCK法觀察細(xì)胞活力,化學(xué)比色法和高效液相色譜法分別觀察心肌細(xì)胞糖酵解關(guān)鍵酶(PK、HK和PFK)活性、肌酸激酶(CK)、乳酸生成,LDH漏出及ATP/ADP生成。 3、免疫印記法及激光共聚焦顯微鏡觀察心肌細(xì)胞微管結(jié)構(gòu)改變后HIF-1α蛋白含量和細(xì)胞內(nèi)分布變化,實(shí)時(shí)定量PCR法檢測(cè)心肌細(xì)胞HIF-1αmRNA表達(dá)變化。 結(jié)果 1、缺氧后早期,體外培養(yǎng)新生大鼠心肌細(xì)胞微管網(wǎng)狀結(jié)構(gòu)破壞,聚合態(tài)α-微管蛋白含量減少,心肌細(xì)胞的活力降低,死亡率升高;穩(wěn)定微管網(wǎng)狀結(jié)構(gòu)可以使細(xì)胞存活率和活性升高。 2、缺氧后早期,體外培養(yǎng)新生大鼠心肌細(xì)胞微管網(wǎng)狀結(jié)構(gòu)破壞引起糖酵解關(guān)鍵酶(PK、HK和PFK)活性降低,代謝終產(chǎn)物乳酸生成減少,細(xì)胞ATP生成減少;而穩(wěn)定微管網(wǎng)狀結(jié)構(gòu)可以在缺氧早期一段時(shí)間內(nèi)升高PK、HK和PFK的活性,促進(jìn)細(xì)胞能量生成。 3、缺氧后早期,體外培養(yǎng)新生大鼠心肌細(xì)胞微管網(wǎng)狀結(jié)構(gòu)破壞使HIF-1α蛋白表達(dá)及入核表達(dá)均減少。微管穩(wěn)定劑和高表達(dá)微管相關(guān)蛋白4可穩(wěn)定缺氧心肌細(xì)胞微管網(wǎng)狀結(jié)構(gòu),上調(diào)HIF-1α蛋白含量及入核表達(dá);微管解聚劑和下調(diào)微管蛋白表達(dá)則可加重缺氧心肌細(xì)胞微管結(jié)構(gòu)破壞,HIF-1α蛋白含量及入核表達(dá)減少更明顯。而且HIF-1α蛋白表達(dá)的增加發(fā)生在轉(zhuǎn)錄后水平。 結(jié)論 微管結(jié)構(gòu)變化通過調(diào)節(jié)HIF-1α可影響缺氧心肌細(xì)胞早期糖酵解。穩(wěn)定微管結(jié)構(gòu)可促進(jìn)HIF-1α入核表達(dá)并提高HIF-1α蛋白含量,提高厭氧糖酵解關(guān)鍵酶活性和能量生成,表明微管結(jié)構(gòu)變化通過調(diào)節(jié)HIF-1α影響缺氧心肌細(xì)胞早期糖酵解,這為臨床改善缺氧早期細(xì)胞能量代謝提供了潛在的治療靶點(diǎn)。
[Abstract]:Purpose Hypoxia is one of the important pathophysiological phenomena in many diseases. Hypoxia inducible factor (HIFs). 1 偽 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 偽. 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 偽-tubulin in cardiomyocytes with high expression of microtubule-associated protein 4 and RNA interference was established. 2. The changes of 偽 -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 偽 protein content and intracellular distribution after microtubule structure changes in cardiomyocytes. The expression of HIF-1 偽 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 偽 -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 偽 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 偽 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 偽 protein and the expression of HIF-1 偽 protein were more obvious, and the increase of HIF-1 偽 protein expression occurred at posttranscriptional level. Conclusion The change of microtubule structure can affect the early glycolysis of hypoxic cardiomyocytes by regulating HIF-1 偽. Stabilizing the microtubule structure can promote the expression of HIF-1 偽 and increase the protein content of HIF-1 偽. 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 偽. This provides a potential therapeutic target for clinical improvement of early anoxic cell energy metabolism.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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