【作者】 刘丽英；

【导师】 董雪松；

【作者基本信息】 中国医科大学 ， 急诊医学（专业学位）， 2021， 硕士

【摘要】 目的:百草枯(paraquat,PQ),化学名称为1,1’-二甲基-4,4’-联吡啶阳离子盐,是目前世界范围内使用量最大的有机杂环类接触性除草剂。急性口服PQ中毒患者,因无特效解毒药物,死亡率非常高。肺脏是PQ中毒的主要受损器官,肺间质纤维化是PQ中毒患者的重要死因。肺泡上皮细胞凋亡过度被认为是肺纤维化发生早期的关键事件之一。PQ诱导的肺泡上皮细胞凋亡涉及到多种凋亡途径,其中线粒体凋亡途径具有重要的作用。研究表明线粒体自噬可抑制细胞凋亡,调高细胞凋亡的阈值,从而改善PQ所致细胞损伤。微小核糖核酸(micro RNAs,miRNAs)是内源性的小非编码核糖核酸,在转录后调节目标基因的表达。miRNAs的发现为多种细胞过程的调控机制提供了新的见解,包括自噬和凋亡。成熟的micro RNA简写成miR。miR-378是近些年来被证实和凋亡发生密切相关的一类miRNA。研究表明,miR-378与众多疾病的发生发展关系密切,涉及到的机制极为复杂。以miR-378调节细胞凋亡为例,在肿瘤细胞中高表达大多可以促进肿瘤细胞凋亡,但在其他疾病中也还包括存在抗凋亡效应,涉及通过抑制Caspase-3和增强线粒体自噬。依据miR-378高表达在神经细胞、骨骼肌、心肌等细胞及组织研究,我们推测PQ损伤II型肺泡上皮细胞(type II of alveolar epithelial cell,AEC2)的过程中,miR-378的高表达在细胞凋亡的过程中有重要意义,并拟在体外细胞实验中予以证实。研究方法:1、miR-378模拟物细胞转染效率的检测:(1)细胞转染:MLE12细胞转染miR-378模拟物及阴性对照物;(2)总RNA提取和实时定量RT-PCR检测miR-378表达。2、miR-378过表达对PQ诱导MLE12细胞凋亡的影响:(1)CCK-8法检测细胞活性;(2)DAPI染色观察细胞核形态的改变。(3)使用Annexin V-FITC/PI双染凋亡试剂盒检测各组细胞的凋亡率;(4)使用Caspases活性检测试剂盒检测Caspase-3/-9活性。3、PQ诱导MLE12细胞凋亡过程中线粒体自噬途径相关指标的检测:(1)MTG和LTR染色,荧光显微镜观察线粒体自噬现象;(2)Western Blotting检测线粒体自噬标志性蛋白LC3和内参β-actin蛋白表达量的变化。结果:1、细胞转染效率的检测:将miR-378模拟物转染到MLE12细胞后,miR-378的表达水平显著增加。2、miR-378过表达对细胞活性的影响:转染miR378-mimics+PQ处理组与未经miR378-mimics转染的MLE12细胞经PQ处理组比较,细胞活性显著增加。3、miR-378过表达对细胞核形态结构的影响:与PQ组相比,miR-378 mimics+PQ组发生核固缩和核碎裂的细胞比例明显降低。4、Mi R-378过表达对细胞凋亡率的影响:与PQ组相比,miR-378 mimics+PQ组细胞凋亡率显著降低。5、Mi R-378过表达对线粒体与溶酶体的融合的影响:使用MTG和LTR在MLE12细胞中共染色,然后通过荧光显微镜分析。与PQ组相比,miR-378 mimics+PQ组线粒体自噬明显增强。6、Mi R-378过表达对Caspase-3、9活性的影响:在PQ+miR-378mimics中,Caspase-3和Caspase-9的活性较PQ组明显降低。7、miR-378过表达增强线粒体自噬标志性蛋白表达:MLE12细胞通过miR-378转染后再接受PQ处理,Western Blotting检测显示线粒体自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ表达增加。结论:在PQ诱导AEC2(MLE12)细胞凋亡中,miR-378过表达可能通过调节线粒体自噬、Caspase-3、9活性水平来抑制细胞凋亡。更多还原

【Abstract】 Objective: Paraquat(PQ),chemically known as 1,1 ’-dimethyl-4,4’-bipyridine cationic salt,is currently the most widely used organic heterocyclic contact herbicide in the world.The mortality rate of patients with acute oral PQ poisoning is very high because there is no specific detoxification drug.Lung is the main damaged organ of PQ poisoning,and pulmonary interstitial fibrosis is an important cause of death in patients with PQ poisoning.The excessive apoptosis of alveolar epithelial cells is considered to be one of the key events in the early development of pulmonary fibrosis.The apoptosis of alveolar epithelial cells induced by PQ involves many apoptotic pathways,among which the mitochondrial apoptotic pathway plays an important role.Other studies have shown that mitochondrial autophagy can inhibit cell apoptosis and increase the threshold of cell apoptosis,thus improving PQ-induced cell damage.Microribonucleic acids(miRNAs)are small,endogenous,non-coding ribonucleic acids that regulate the expression of target genes after transcription.The discovery of miRNAs has provided new insights into the regulatory mechanisms of multiple cellular processes,including autophagy and apoptosis.Mature micro RNAs are abbreviated as miR.Mi R-378 is a class of miRNAs that have been proved to be closely related to apoptosis in recent years.Studies have shown that miR-378 is closely related to the occurrence and development of many diseases,and the mechanisms involved are extremely complex.Taking the regulation of apoptosis of miR-378 as an example,the high expression of miR-378 in tumor cells can mostly promote the apoptosis of tumor cells,but there are also anti-apoptotic effects in other diseases,involving inhibition of caspase-3 and enhancement of mitochondrial autophagy.According to the studies on the high expression of miR-378 in nerve cells,skeletal muscle,cardiac muscle and other cells and tissues,we speculated that the high expression of miR-378 plays an important role in the process of apoptosis of Type II of alveolar epithelial cells(AEC2)during PQ injury,and it is planned to be confirmed in vitro cell experiments.Methods: 1.Detection of cell transfection efficiency of miR-378 mimics:(1)Cell transfection: MLE12 cells were transfected with miR-378 mimics and negative control;(2)Total RNA extraction and real-time quantitative RT-PCR were used to detect the expression of miR-378.2.Effects of miR-378 overexpression on PQ-induced apoptosis of MLE12 cells:(1)CCK-8 assay was used to detect cell activity;(2)The changes of nuclear morphology were observed by DAPI staining;(3)The apoptosis rate of cells in each group was detected by Annexin V-FITC/PI double-staining apoptosis kit;(4)The activity of caspase-3 /-9 was detected with the Caspases activity detection kit;3.Detection of indicators related to mitochondrial autophagy pathway in the process of PQ-induced apoptosis of Mle12 cells:(1)Mitochondrial autophagy was observed by fluorescence microscopy with MTG and LTR staining;(2)The expression levels of LC3 and β-actin were detected by Western Blotting.Results: 1.Detection of cell transfection efficiency: After transfection of miR-378 mimics into MLE12 cells,the expression level of miR-378 was significantly increased.2.Effects of miR-378 overexpression on cell activity: The cell viability of MLE12 cells transfected with miR378-mimics+PQ was significantly increased compared with that of MLE12 cells transfected with miR378-mimics+PQ.3.Effects of miR-378 overexpression on nuclear morphology and structure:Compared with the PQ group,the proportion of cells with nuclear pyknosis and fragmentation in the miR-378 mimics +PQ group was significantly lower.4.Effects of miR-378 overexpression on apoptosis rate: Compared with the PQ group,the apoptosis rate of miR-378 mimics +PQ group was significantly reduced.5.Effects of miR-378 overexpression on mitochondria-lysosome fusion:MLE12 cells were co-stained with MTG and LTR,and then analyzed by fluorescence microscopy.Compared with the PQ group,mitochondrial autophagy was significantly enhanced in the miR-378 mimics +PQ group.6.Effects of miR-378 overexpression on caspase-3 and 9 activities: The activities of caspase-3 and caspase-9 in PQ+miR-378 mimics were significantly lower than those in PQ group.7.Overexpression of miR-378 enhanced the expression of mitochondrial autophagy signature proteins: MLE12 cells by miR-378 transfection accept PQ again after processing,Western Blotting test shows that the mitochondrial autophagy related proteins LC3-Ⅱ / LC3-Ⅰ expression increases.Conclusion: In PQ-induced apoptosis of AEC2(MLE12)cells,overexpression of miR-378 may inhibit apoptosis by regulating the activity levels of mitochondrial autophagy,caspase-3 and 9.更多还原