本文選題：脂多糖 + 自吞噬�。� 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：膿毒血癥是以多器官功能衰竭和心血管抑制為特征的全身性炎性反應(yīng)綜合癥,臨床研究證明心臟功能抑制顯著增加膿毒血癥病人的死亡率。在細(xì)胞水平的研究發(fā)現(xiàn)心肌細(xì)胞凋亡和能量代謝障礙是膿毒血癥中心血管功能衰竭的重要機(jī)制。 在膿毒血癥心肌細(xì)胞中發(fā)現(xiàn)了自吞噬體增加、線粒體損傷以及內(nèi)質(zhì)網(wǎng)應(yīng)激的現(xiàn)象,并且有研究發(fā)現(xiàn)這三者之間存在互為因果的關(guān)系。自吞噬是真核細(xì)胞內(nèi)進(jìn)化過(guò)程高保守的分解代謝過(guò)程,包括降解長(zhǎng)壽蛋白和清除老化受損細(xì)胞器,最終的降解產(chǎn)物參與新的能量代謝。近年來(lái)研究發(fā)現(xiàn),自吞噬選擇性地清除受損的線粒體有利于細(xì)胞穩(wěn)態(tài)的維持,促進(jìn)應(yīng)激狀態(tài)下的細(xì)胞存活。線粒體是細(xì)胞內(nèi)能量代謝的主要場(chǎng)所,并參與調(diào)節(jié)細(xì)胞凋亡途徑的調(diào)節(jié)。線粒體的質(zhì)量控制依賴受損／老化線粒體的修復(fù)／清除和線粒體生物合成之間的動(dòng)態(tài)平衡,但是在膿毒血癥心肌細(xì)胞中自吞噬對(duì)線粒體再生的影響還需進(jìn)一步的研究。雖然很多研究已經(jīng)證明未內(nèi)質(zhì)網(wǎng)應(yīng)激的適應(yīng)性未折疊蛋白反應(yīng)參與了自吞噬的發(fā)生,但這個(gè)觀點(diǎn)在膿毒血癥心肌細(xì)胞中未見(jiàn)報(bào)道。 我們通過(guò)建立HL-1心肌細(xì)胞膿毒血癥模型,研究自吞噬的表達(dá)、發(fā)生和自吞噬抗細(xì)胞凋亡的保護(hù)機(jī)制,為膿毒血癥心臟功能衰竭提供臨床治療靶點(diǎn)。 ■第一部分脂多糖誘導(dǎo)自吞噬對(duì)HL-1心肌細(xì)胞凋亡的影響 目的：研究脂多糖誘導(dǎo)HL-1心肌細(xì)胞自吞噬表達(dá)及自吞噬對(duì)心肌細(xì)胞的保護(hù)作用。 方法：建立HL-1心肌細(xì)胞膿毒癥模型,脂多糖以1ug/ml終濃度干預(yù)HL-1心肌細(xì)胞。應(yīng)用蛋白免疫印跡方法觀察脂多糖干預(yù)HL-1心肌細(xì)胞2、4、8、16、24h的LC3Ⅱ蛋白的表達(dá)時(shí)程；在脂多糖干預(yù)4和24h,通過(guò)透射電子顯微鏡和共聚焦顯微鏡觀察自吞噬體的形成,時(shí)時(shí)定量聚合酶鏈反應(yīng)檢測(cè)ATG5和ATG7mRNA的表達(dá)評(píng)估自吞噬活性,通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。3-甲基嘌呤(3-MA)和納巴霉素(Rap)預(yù)處理48h后,分別觀察脂多糖干預(yù)4和24h心肌細(xì)胞凋亡和LC3Ⅱ蛋白表達(dá)。結(jié)果：脂多糖干預(yù)HL-1心肌細(xì)胞2h后LC3Ⅱ開(kāi)始上調(diào),4h達(dá)到高峰,24h減弱；共聚焦顯微鏡顯示,脂多糖干預(yù)4h后出現(xiàn)綠色熒光斑點(diǎn)物質(zhì)出現(xiàn)聚集,24h減弱；電子顯微鏡顯示在脂多糖干預(yù)4h,出現(xiàn)了雙膜或多膜結(jié)構(gòu)的自吞噬體,而在24h減少。ATG5和ATG7mRNA表達(dá)在脂多糖干預(yù)后4h上調(diào),同樣在24h下降。3-MA預(yù)處理后,誘導(dǎo)了脂多糖干預(yù)4h的細(xì)胞凋亡。Rap預(yù)處理后,抑制了脂多糖在24h誘導(dǎo)的細(xì)胞凋亡。結(jié)論：脂多糖在HL-1心肌細(xì)胞誘導(dǎo)了自吞噬,表現(xiàn)為早期增強(qiáng),而晚期表現(xiàn)為下降。自吞噬在膿毒癥中表現(xiàn)為細(xì)胞保護(hù)作用。 ■第二部分脂多糖誘導(dǎo)自吞噬對(duì)HL-1心肌細(xì)胞線粒體功能影響的研究 目的：研究膿毒血癥心肌細(xì)胞中自吞噬線粒體功能和線粒體再生的影響。方法：建立HL-1心肌細(xì)胞內(nèi)毒素血癥模型。在脂多糖干預(yù)24h后,通過(guò)電子顯微鏡、流式細(xì)胞儀和時(shí)時(shí)定量聚合酶鏈反應(yīng)技術(shù)觀察線粒體面積和光密度,線粒體膜電位,線粒體再生基因Tfam和PGC-1a的變化評(píng)估線粒體功能。脂多糖干預(yù)前,3-甲基嘌呤(3-MA)和納巴霉素(Rap)預(yù)處理48h,觀察線粒體功能及線粒體再生的變化。結(jié)果：HL-1心肌細(xì)胞在脂多糖干預(yù)24h后,線粒體面積和光密度增加,線粒體膜電位下降,并伴隨線粒體再生基因Tfam和PGC-1amRNA表達(dá)下調(diào)。3-MA預(yù)處理下調(diào)自吞噬的表達(dá),加重了脂多糖對(duì)線粒體功能的損傷和進(jìn)一步抑制了線粒體再生基因的表達(dá)。納巴霉素預(yù)處理,上調(diào)自吞噬的表達(dá),減輕了脂多糖對(duì)線粒體功能的損傷,同時(shí)線粒體再生功能上調(diào)。結(jié)論：脂多糖在HL-1心肌細(xì)胞導(dǎo)致了線粒體超微結(jié)構(gòu)的損傷和線粒體膜電位下降,抑制了線粒體的再生功能。抑制自吞噬,加重了脂多糖對(duì)線粒體的損傷,上調(diào)自吞噬,減輕了脂多糖對(duì)線粒體功能的損傷。自吞噬在HL-1心肌細(xì)胞內(nèi)毒素血癥中對(duì)線粒體功能起到保護(hù)作用。 ■第三部分脂多糖在HL-1心肌細(xì)胞誘導(dǎo)自吞噬發(fā)生機(jī)制的研究 目的：研究在HL-1心肌細(xì)胞中脂多糖是否通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)自吞噬。方法：建立HL-1心肌細(xì)胞脂多糖內(nèi)毒血癥模型。通過(guò)時(shí)時(shí)定量聚合酶鏈反應(yīng)檢測(cè)脂多糖干預(yù)4、24h和對(duì)照組GRP78和IRElamRNA表達(dá),觀察脂多糖誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激的變化。衣霉素(Tm)和�；敲撗跄懰�(TUDCA)預(yù)處理后,通過(guò)時(shí)時(shí)定量聚合酶鏈反應(yīng)、蛋白免疫印跡和共聚焦顯微鏡觀察脂多糖干預(yù)HL-1心肌細(xì)胞4h后,GRP78、IREla、ATG5和ATG7mRNA,LC3II蛋白印跡和綠色LC3Ⅱ熒光顆粒的表達(dá)評(píng)估的內(nèi)質(zhì)網(wǎng)應(yīng)激和自吞噬變化的關(guān)系。結(jié)果：脂多糖在HL-1心肌細(xì)胞中誘導(dǎo)了內(nèi)質(zhì)網(wǎng)應(yīng)激,與對(duì)照組比較,GRP78和IRElamRNA在干預(yù)后4h表達(dá)上調(diào),24h表達(dá)下降。與對(duì)照組比較,衣霉素誘導(dǎo)了GRP78和IRElamRNA、LC3II蛋白印跡和綠色LC3Ⅱ熒光表達(dá)顆粒上調(diào)；與脂多糖干預(yù)4h組比較,衣霉素增強(qiáng)了脂多糖誘導(dǎo)的GRP78和IREla的表達(dá),同時(shí)進(jìn)一步增加了LC3Ⅱ蛋白印跡和綠色LC3Ⅱ熒光顆粒的表達(dá)。�；敲撗跄懰犷A(yù)處理減輕了脂多糖誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激,同時(shí)自吞噬的表達(dá)下降。結(jié)論：脂多糖在HL-1心肌細(xì)胞中誘導(dǎo)了內(nèi)質(zhì)網(wǎng)應(yīng)激和自吞噬的表達(dá)。脂多糖在H1-1心肌細(xì)胞誘導(dǎo)自吞噬是通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激實(shí)現(xiàn)的。
[Abstract]:Sepsis is a systemic inflammatory response syndrome characterized by multiple organ failure and cardiovascular inhibition. Clinical studies have demonstrated that cardiac function inhibition significantly increases the mortality of patients with sepsis. Cell apoptosis and energy metabolism disorders are important mechanisms of cardiac failure in sepsis. System.
The increase of autophago, mitochondrial damage and endoplasmic reticulum stress were found in septic cardiomyocytes, and there was a study of the relationship between the three. Autophagy was a highly conservative metabolic process in eukaryotic cell evolution, including the reduction of longevity protein and the elimination of aging damaged organelles. The degradation products are involved in the new energy metabolism. In recent years, it has been found that autophagy selectively scavenging damaged mitochondria is beneficial to the maintenance of cell homeostasis and promotes cell survival in stress state. Mitochondria are the main sites of intracellular energy metabolism and regulate the regulation of cell withering pathways. Mitochondrial quality control dependence The dynamic balance between damaged / aging mitochondria repair / scavenging and mitochondrial biosynthesis, but the effect of autophagy on mitochondrial regeneration in septic myocardial cells needs further study. Although many studies have shown that the adaptive unfolding of unfolded protein reaction in the non endoplasmic reticulum stress is involved in the occurrence of autophagy, but This view has not been reported in septic cardiomyocytes.
We have established the HL-1 cardiomyocyte sepsis model to study the protective mechanism of autophagy expression, occurrence and autophagy, and to provide clinical therapeutic targets for cardiac failure of sepsis.
Part one effect of lipopolysaccharide induced autophagy on the apoptosis of HL-1 cardiomyocytes
Objective: To study the autophagic expression of lipopolysaccharide induced HL-1 cardiomyocytes and the protective effect of autophagy on cardiomyocytes.
Methods: the HL-1 cardiac myocytic sepsis model was established, and the lipopolysaccharide interfered with the HL-1 myocardial cells at the end concentration of 1ug/ml. The expression of LC3 II protein was observed by lipopolysaccharide in HL-1 cardiac myocyte 2,4,8,16,24h by Western blot. The autophagy was observed in 4 and 24h by lipopolysaccharide, and the autophagy was observed through the transmission electron microscope and confocal microscope. The expression of ATG5 and ATG7mRNA was measured by quantitative polymerase chain reaction (PCR) to evaluate the autophagy activity. After the apoptosis of.3- methyl purine (3-MA) and napimycin (Rap) was pretreated by flow cytometry, the expression of lipopolysaccharide intervention in 4 and 24h cardiomyocytes and the expression of LC3 II protein were observed respectively. Results: lipopolysaccharide intervened HL-1 myocardium. After 2h, LC3 II began to rise, 4H reached its peak, and 24h weakened. Confocal microscopy showed that the green fluorescent spots were aggregated and 24h weakened after the intervention of 4h, and the electron microscope showed that lipopolysaccharide intervened 4h, and there was a double or multi membrane autophago, while 24h reduced.ATG5 and ATG7mRNA expression in lipopolysaccharide. The prognosis of 4H was up-regulated, and after 24h decreased.3-MA preconditioning, it induced lipopolysaccharide to interfere with apoptosis of 4H by.Rap preconditioning, and inhibited the apoptosis induced by 24h. Conclusion: lipopolysaccharide induced autophagy in HL-1 cardiomyocytes, showing early enhancement, but decreased in late stage. Autophagy is a cell in sepsis. Protective effect.
The effect of lipopolysaccharide induced autophagy on mitochondrial function of HL-1 cardiomyocytes in part second
Objective: To study the effect of autophagic mitochondrial function and mitochondrial regeneration in septic cardiomyocytes. Methods: to establish a HL-1 cardiomyocyte endotoxemia model. After 24h was intervened in lipopolysaccharide, the mitochondrial area and light density, and mitochondrial membrane electricity was observed by electron microscopy, flow cytometry and time quantitative polymerase chain reaction. The mitochondrial function was evaluated by the changes of mitochondrial regenerative gene Tfam and PGC-1a. Before the intervention of lipopolysaccharide, 3- methyl purine (3-MA) and napimycin (Rap) were pretreated with 48h to observe the changes in mitochondrial function and mitochondrial regeneration. Results: the mitochondrial area and light density increased and the mitochondrial membrane potential decreased after the intervention of 24h in the lipopolysaccharide. The expression of autophagy was down regulated by the down-regulation of the mitochondrial regeneration gene Tfam and PGC-1amRNA, which aggravated the damage of the mitochondrial function and further inhibition of the expression of the mitochondrial regenerative gene. Conclusion: the ultrastructural damage of mitochondria and the decrease of mitochondrial membrane potential caused by lipopolysaccharide in HL-1 cardiomyocytes inhibit the regeneration of mitochondria. Inhibition of autophagy, aggravated the damage of lipopolysaccharide to mitochondria, up regulation of autophagy, and alleviated the damage to mitochondrial function. Self phagocytosis at HL-1 Myocardial cell endotoxemia plays a protective role in mitochondrial function.
The third part of LPS induces autophagy in HL-1 cardiomyocytes.
Objective: To investigate whether lipopolysaccharide induced autophagy by endoplasmic reticulum stress in HL-1 cardiomyocytes. Methods: to establish a model of lipopolysaccharide endotoxemia in HL-1 cardiomyocytes. The expression of GRP78 and IRElamRNA in 4,24h and control groups was detected by time quantitative polymerase chain reaction (PCR), and the changes of endoplasmic reticulum stress induced by lipopolysaccharide were observed. After pretreatment with Tm and Taurodeoxycholic acid (TUDCA), the relationship between endoplasmic reticulum stress and autophagy changes was observed by the quantitative polymerase chain reaction, Western blot and confocal microscopy in the intervention of 4h, GRP78, IREla, ATG5 and ATG7mRNA, and the expression of LC3II and green LC3 II fluorescent particles in HL-1 cardiomyocytes. Results: lipopolysaccharide induced endoplasmic reticulum stress in HL-1 cardiomyocytes. Compared with the control group, the expression of GRP78 and IRElamRNA was up and 24h expression decreased after intervention. Compared with the control group, yimycin induced GRP78 and IRElamRNA, LC3II blot and green LC3 II fluorescent expression particles up up, compared with the lipopolysaccharide intervention 4H group, ycomycin. The expression of GRP78 and IREla induced by lipopolysaccharide was enhanced, and the expression of LC3 II blot and green LC3 II fluorescent particles was further increased. The preconditioning of taurocholic acid alleviated the endoplasmic reticulum stress induced by lipopolysaccharide and decreased the expression of autophagy. Conclusion: lipopolysaccharide induced endoplasmic reticulum stress in HL-1 cardiomyocytes and Autophagy. LPS induced autophagy in H1-1 cardiomyocytes was achieved through endoplasmic reticulum stress.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

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相關(guān)碩士學(xué)位論文 前1條

1 楊新蔚;重組可溶性MICA蛋白對(duì)NK細(xì)胞NKG2D受體表達(dá)的影響[D];蘭州大學(xué);2012年

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本文編號(hào)：1890227