内质网应激诱导自吞噬对脂多糖诱导HL-1心肌细胞损伤保护机制的研究

发布时间：2018-05-15 00:42

本文选题：脂多糖 + 自吞噬　；参考：《华中科技大学》2012年博士论文

【摘要】：脓毒血症是以多器官功能衰竭和心血管抑制为特征的全身性炎性反应综合症,临床研究证明心脏功能抑制显著增加脓毒血症病人的死亡率。在细胞水平的研究发现心肌细胞凋亡和能量代谢障碍是脓毒血症中心血管功能衰竭的重要机制。在脓毒血症心肌细胞中发现了自吞噬体增加、线粒体损伤以及内质网应激的现象,并且有研究发现这三者之间存在互为因果的关系。自吞噬是真核细胞内进化过程高保守的分解代谢过程,包括降解长寿蛋白和清除老化受损细胞器,最终的降解产物参与新的能量代谢。近年来研究发现,自吞噬选择性地清除受损的线粒体有利于细胞稳态的维持,促进应激状态下的细胞存活。线粒体是细胞内能量代谢的主要场所,并参与调节细胞凋亡途径的调节。线粒体的质量控制依赖受损／老化线粒体的修复／清除和线粒体生物合成之间的动态平衡,但是在脓毒血症心肌细胞中自吞噬对线粒体再生的影响还需进一步的研究。虽然很多研究已经证明未内质网应激的适应性未折叠蛋白反应参与了自吞噬的发生,但这个观点在脓毒血症心肌细胞中未见报道。我们通过建立HL-1心肌细胞脓毒血症模型,研究自吞噬的表达、发生和自吞噬抗细胞凋亡的保护机制,为脓毒血症心脏功能衰竭提供临床治疗靶点。 ■第一部分脂多糖诱导自吞噬对HL-1心肌细胞凋亡的影响目的：研究脂多糖诱导HL-1心肌细胞自吞噬表达及自吞噬对心肌细胞的保护作用。方法：建立HL-1心肌细胞脓毒症模型,脂多糖以1ug/ml终浓度干预HL-1心肌细胞。应用蛋白免疫印迹方法观察脂多糖干预HL-1心肌细胞2、4、8、16、24h的LC3Ⅱ蛋白的表达时程；在脂多糖干预4和24h,通过透射电子显微镜和共聚焦显微镜观察自吞噬体的形成,时时定量聚合酶链反应检测ATG5和ATG7mRNA的表达评估自吞噬活性,通过流式细胞仪检测细胞凋亡。3-甲基嘌呤(3-MA)和纳巴霉素(Rap)预处理48h后,分别观察脂多糖干预4和24h心肌细胞凋亡和LC3Ⅱ蛋白表达。结果：脂多糖干预HL-1心肌细胞2h后LC3Ⅱ开始上调,4h达到高峰,24h减弱；共聚焦显微镜显示,脂多糖干预4h后出现绿色荧光斑点物质出现聚集,24h减弱；电子显微镜显示在脂多糖干预4h,出现了双膜或多膜结构的自吞噬体,而在24h减少。ATG5和ATG7mRNA表达在脂多糖干预后4h上调,同样在24h下降。3-MA预处理后,诱导了脂多糖干预4h的细胞凋亡。Rap预处理后,抑制了脂多糖在24h诱导的细胞凋亡。结论：脂多糖在HL-1心肌细胞诱导了自吞噬,表现为早期增强,而晚期表现为下降。自吞噬在脓毒症中表现为细胞保护作用。 ■第二部分脂多糖诱导自吞噬对HL-1心肌细胞线粒体功能影响的研究目的：研究脓毒血症心肌细胞中自吞噬线粒体功能和线粒体再生的影响。方法：建立HL-1心肌细胞内毒素血症模型。在脂多糖干预24h后,通过电子显微镜、流式细胞仪和时时定量聚合酶链反应技术观察线粒体面积和光密度,线粒体膜电位,线粒体再生基因Tfam和PGC-1a的变化评估线粒体功能。脂多糖干预前,3-甲基嘌呤(3-MA)和纳巴霉素(Rap)预处理48h,观察线粒体功能及线粒体再生的变化。结果：HL-1心肌细胞在脂多糖干预24h后,线粒体面积和光密度增加,线粒体膜电位下降,并伴随线粒体再生基因Tfam和PGC-1amRNA表达下调。3-MA预处理下调自吞噬的表达,加重了脂多糖对线粒体功能的损伤和进一步抑制了线粒体再生基因的表达。纳巴霉素预处理,上调自吞噬的表达,减轻了脂多糖对线粒体功能的损伤,同时线粒体再生功能上调。结论：脂多糖在HL-1心肌细胞导致了线粒体超微结构的损伤和线粒体膜电位下降,抑制了线粒体的再生功能。抑制自吞噬,加重了脂多糖对线粒体的损伤,上调自吞噬,减轻了脂多糖对线粒体功能的损伤。自吞噬在HL-1心肌细胞内毒素血症中对线粒体功能起到保护作用。 ■第三部分脂多糖在HL-1心肌细胞诱导自吞噬发生机制的研究目的：研究在HL-1心肌细胞中脂多糖是否通过内质网应激诱导自吞噬。方法：建立HL-1心肌细胞脂多糖内毒血症模型。通过时时定量聚合酶链反应检测脂多糖干预4、24h和对照组GRP78和IRElamRNA表达,观察脂多糖诱导内质网应激的变化。衣霉素(Tm)和牛磺脱氧胆酸(TUDCA)预处理后,通过时时定量聚合酶链反应、蛋白免疫印迹和共聚焦显微镜观察脂多糖干预HL-1心肌细胞4h后,GRP78、IREla、ATG5和ATG7mRNA,LC3II蛋白印迹和绿色LC3Ⅱ荧光颗粒的表达评估的内质网应激和自吞噬变化的关系。结果：脂多糖在HL-1心肌细胞中诱导了内质网应激,与对照组比较,GRP78和IRElamRNA在干预后4h表达上调,24h表达下降。与对照组比较,衣霉素诱导了GRP78和IRElamRNA、LC3II蛋白印迹和绿色LC3Ⅱ荧光表达颗粒上调；与脂多糖干预4h组比较,衣霉素增强了脂多糖诱导的GRP78和IREla的表达,同时进一步增加了LC3Ⅱ蛋白印迹和绿色LC3Ⅱ荧光颗粒的表达。牛磺脱氧胆酸预处理减轻了脂多糖诱导的内质网应激,同时自吞噬的表达下降。结论：脂多糖在HL-1心肌细胞中诱导了内质网应激和自吞噬的表达。脂多糖在H1-1心肌细胞诱导自吞噬是通过内质网应激实现的。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】