慢性炎症影响气道上皮细胞纤毛摆动频率的机制研究

发布时间：2018-03-10 21:08

本文选题：纤毛摆动频率　切入点：慢性炎症　出处：《华中科技大学》2013年硕士论文　论文类型：学位论文

【摘要】：慢性气道炎症是促进呼吸系统疾病发生发展的常见原因之一，而在慢性气道炎症中，往往会出现纤毛摆动频率的下降，使得气道出现疾患。本研究为了探讨慢性炎症时气道上皮细胞纤毛摆动频率下降的机制，通过大鼠原代气道上皮细胞培养，高速显微摄像，Elisa检测蛋白，JC-1探针检测膜电位以及DCFHDA检测活性氧几种方法，分别检测细胞纤毛摆动频率，LTB_4含量，线粒体膜电位以及活性氧几种指标。发现大鼠气道上皮细胞在受到香烟提取物+脂多糖刺激3h后，活性氧产生增加，，且线粒体膜电位下降，细胞胆碱能亢进导致LTB_4含量上升，细胞纤毛摆动频率下降，有显著性差异（p0.05）。在使用胆碱能抑制剂异丙托溴铵和香烟提取物+脂多糖共同作用后，细胞内LTB_4含量下降，纤毛摆动频率恢复，与香烟提取物+脂多糖组相比有显著差异（p0.05）。同时在使用H_2O_2刺激细胞引起的胆碱能功能亢进和香烟提取物+脂多糖刺激相似，无显著变化（p0.05）。而在使用过氧化物清除剂PEG-SOD与PEG-catalase和香烟提取物+脂多糖共同刺激后活性氧浓度以及LTB_4含量均下降，有明显差异（p0.05）。由此说明，在气道上皮细胞慢性炎症时，细胞纤毛摆动频率的下降是由于线粒体损伤导致活性氧产生增多进而引起细胞胆碱能亢进所造成的。目的本实验旨在通过大鼠气道上皮细胞原代培养后使用香烟提取物+脂多糖刺激诱导细胞慢性炎症，探讨在慢性气道炎症时引起细胞纤毛摆动频率下降的机制。方法 1．根据Davidson等方法加以改进，使用大鼠气道环贴块法培养大鼠气道上皮细胞。. 2．利用高速显微摄像技术记录纤毛上皮细胞的高速纤毛摆动情况，并且利用图像灰度分析软件对纤毛摆动频率进行分析。 3.利用Elisa检测细胞上清中分泌的LTB_4蛋白的含量。 4.利用JC-1探针检测细胞线粒体膜电位。 5.利用DCFHDA染料检测细胞活性氧。结果大鼠气道上皮细胞在受到香烟提取物+脂多糖刺激3h后，活性氧浓度增加，且线粒体膜电位下降，细胞胆碱能功能亢进导致LTB_4含量上升，细胞纤毛摆动频率下降，有显著性差异（p0.05）。而活性氧的增多导致胆碱能亢进，进而导致纤毛摆动频率下降，在使用胆碱能抑制剂异丙托溴铵和香烟提取物+脂多糖共同作用后，细胞胆碱能功能亢进逆转导致LTB_4含量下降，纤毛摆动频率回升，有显著差异（p0.05）。同时在使用H_2O_2刺激细胞时产生LTB_4的量和香烟提取物+脂多糖刺激相似，无显著变化（p0.05）。而在使用活性氧清除剂PEG-SOD与PEG-catalase和香烟脂多糖共同刺激后活性氧浓度以及LTB_4含量均下降，有明显差异（p0.05）。结论本实验研究成功复制了气道上皮细胞纤毛摆动的模型，阐述了慢性炎症时气道上皮细胞纤毛摆动频率下降的机制；由本实验研究结果说明，在气道上皮细胞慢性炎症时，细胞纤毛摆动频率的下降是由于线粒体损伤导致活性氧增多进而引起了细胞胆碱能亢进所造成的；同时也为研究纤毛摆动以及相关机制奠定理论基础。
[Abstract]:Chronic airway inflammation is a common cause of promoting the occurrence and development of respiratory diseases, and in chronic airway inflammation, decreased often cilia beat frequency, the airway diseases occur. This research is to investigate the mechanism of chronic inflammation of airway epithelial cells of cilia swing frequency decreased, the primary rat airway epithelial cell culture, high speed microscopic imaging, detection of Elisa protein, JC-1 probe for detection of membrane potential and reactive oxygen species DCFHDA detection methods were used to examine cell ciliary beat frequency, LTB_4 content, mitochondrial membrane potential and reactive oxygen species. Several indicators found in airway epithelial cells of rat by cigarette smoke extract + LPS after 3H stimulation increased the generation of reactive oxygen species, and mitochondrial membrane potential decreased cell cholinergic hyperactivity leads to increased LTB_4 content, cell ciliary beat frequency decreased, there was significant difference (P0.05). In the use of cholinergic Inhibitors of ipratropium bromide and common effect of cigarette smoke extract + lipopolysaccharide, decreased LTB_4 content in the cell, restore ciliary beat frequency, there are significant differences compared with cigarette smoke extract + LPS group (P0.05). At the same time in the use of H_2O_2 cells induced by stimulation of cholinergic function hyperfunction and CSE + LPS Stimulation, no significant change (P0.05). While the concentration of active oxygen and LTB_4 content decreased in the use of peroxide scavenger PEG-SOD and PEG-catalase and CSE + LPS stimulation, there was significant difference (P0.05). This shows that chronic inflammation in the airway epithelial cells, decrease cell ciliary beat frequency is due to the number of active oxygen and cause cholinergic cells hyperthyroidism caused by mitochondrial damage.
objective
The aim of this study is to induce chronic inflammation in rats after primary culture of rat airway epithelial cells, using cigarette extract and lipopolysaccharide stimulation, and to explore the mechanism of cell ciliary oscillation frequency decline during chronic airway inflammation.
Method
1. the rat airway epithelial cells were cultured by the method of Davidson and other methods.
2., we use high-speed microphotography to record ciliated epithelial cell's high-speed cilia swing, and analyze the cilia oscillation frequency by image gray analysis software.
3. the content of LTB_4 protein secreted in cell supernatant was detected by Elisa.
4. the cell mitochondrial membrane potential was detected by JC-1 probe.
5. using DCFHDA dye to detect cell reactive oxygen species.
Airway epithelial cells of rat by cigarette smoke extract + LPS stimulated 3H activity, increase oxygen concentration, and decreased the mitochondrial membrane potential, cell cholinergic function hyperfunction caused the increase of LTB_4 in cell ciliary beat frequency decreased, there was significant difference (P0.05). And the increase of reactive oxygen leads to cholinergic hyperactivity, which led to the ciliotoxicity the frequency of decline in the use of cholinergic inhibitors of ipratropium bromide and common effect of cigarette smoke extract after LPS + cells, cholinergic function hyperfunction reversal resulted in decrease of LTB_4 content, ciliary beat frequency rise, there are significant differences (P0.05). At the same time in the use of H_2O_2 stimulated cells generated when the amount of LTB_4 and cigarette smoke extract + LPS stimulation similar. No significant change (P0.05). And in the use of active oxygen scavenger PEG-SOD and PEG-catalase costimulatory activity after lipopolysaccharide and cigarette oxygen concentration and LTB_4 content decreased, Ming Significant difference (P0.05).
This study successfully established airway epithelial cells ciliotoxicity model, expounds the mechanism of chronic inflammation of airway epithelial cells of cilia swing frequency decreased; illustrated by the results of the experiment, chronic inflammation in the airway epithelial cells, decrease cell ciliary beat frequency is caused by ROS and cause cell cholinergic hyperactivity due to mitochondrial damage; but also lay the theoretical foundation for the study of ciliary movement and the related mechanism.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562

【共引文献】