本文選題：纖毛擺動頻率　切入點：慢性炎癥　出處：《華中科技大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：慢性氣道炎癥是促進(jìn)呼吸系統(tǒng)疾病發(fā)生發(fā)展的常見原因之一，而在慢性氣道炎癥中，往往會出現(xiàn)纖毛擺動頻率的下降，使得氣道出現(xiàn)疾患。本研究為了探討慢性炎癥時氣道上皮細(xì)胞纖毛擺動頻率下降的機制，通過大鼠原代氣道上皮細(xì)胞培養(yǎng)，高速顯微攝像，Elisa檢測蛋白，JC-1探針檢測膜電位以及DCFHDA檢測活性氧幾種方法，分別檢測細(xì)胞纖毛擺動頻率，LTB_4含量，線粒體膜電位以及活性氧幾種指標(biāo)。發(fā)現(xiàn)大鼠氣道上皮細(xì)胞在受到香煙提取物+脂多糖刺激3h后，活性氧產(chǎn)生增加，，且線粒體膜電位下降，細(xì)胞膽堿能亢進(jìn)導(dǎo)致LTB_4含量上升，細(xì)胞纖毛擺動頻率下降，有顯著性差異（p0.05）。在使用膽堿能抑制劑異丙托溴銨和香煙提取物+脂多糖共同作用后，細(xì)胞內(nèi)LTB_4含量下降，纖毛擺動頻率恢復(fù)，與香煙提取物+脂多糖組相比有顯著差異（p0.05）。同時在使用H_2O_2刺激細(xì)胞引起的膽堿能功能亢進(jìn)和香煙提取物+脂多糖刺激相似，無顯著變化（p0.05）。而在使用過氧化物清除劑PEG-SOD與PEG-catalase和香煙提取物+脂多糖共同刺激后活性氧濃度以及LTB_4含量均下降，有明顯差異（p0.05）。由此說明，在氣道上皮細(xì)胞慢性炎癥時，細(xì)胞纖毛擺動頻率的下降是由于線粒體損傷導(dǎo)致活性氧產(chǎn)生增多進(jìn)而引起細(xì)胞膽堿能亢進(jìn)所造成的。 目的 本實驗旨在通過大鼠氣道上皮細(xì)胞原代培養(yǎng)后使用香煙提取物+脂多糖刺激誘導(dǎo)細(xì)胞慢性炎癥，探討在慢性氣道炎癥時引起細(xì)胞纖毛擺動頻率下降的機制。 方法 1．根據(jù)Davidson等方法加以改進(jìn)，使用大鼠氣道環(huán)貼塊法培養(yǎng)大鼠氣道上皮細(xì)胞。. 2．利用高速顯微攝像技術(shù)記錄纖毛上皮細(xì)胞的高速纖毛擺動情況，并且利用圖像灰度分析軟件對纖毛擺動頻率進(jìn)行分析。 3.利用Elisa檢測細(xì)胞上清中分泌的LTB_4蛋白的含量。 4.利用JC-1探針檢測細(xì)胞線粒體膜電位。 5.利用DCFHDA染料檢測細(xì)胞活性氧。結(jié)果 大鼠氣道上皮細(xì)胞在受到香煙提取物+脂多糖刺激3h后，活性氧濃度增加，且線粒體膜電位下降，細(xì)胞膽堿能功能亢進(jìn)導(dǎo)致LTB_4含量上升，細(xì)胞纖毛擺動頻率下降，有顯著性差異（p0.05）。而活性氧的增多導(dǎo)致膽堿能亢進(jìn)，進(jìn)而導(dǎo)致纖毛擺動頻率下降，在使用膽堿能抑制劑異丙托溴銨和香煙提取物+脂多糖共同作用后，細(xì)胞膽堿能功能亢進(jìn)逆轉(zhuǎn)導(dǎo)致LTB_4含量下降，纖毛擺動頻率回升，有顯著差異（p0.05）。同時在使用H_2O_2刺激細(xì)胞時產(chǎn)生LTB_4的量和香煙提取物+脂多糖刺激相似，無顯著變化（p0.05）。而在使用活性氧清除劑PEG-SOD與PEG-catalase和香煙脂多糖共同刺激后活性氧濃度以及LTB_4含量均下降，有明顯差異（p0.05）。結(jié)論 本實驗研究成功復(fù)制了氣道上皮細(xì)胞纖毛擺動的模型，闡述了慢性炎癥時氣道上皮細(xì)胞纖毛擺動頻率下降的機制；由本實驗研究結(jié)果說明，在氣道上皮細(xì)胞慢性炎癥時，細(xì)胞纖毛擺動頻率的下降是由于線粒體損傷導(dǎo)致活性氧增多進(jìn)而引起了細(xì)胞膽堿能亢進(jìn)所造成的；同時也為研究纖毛擺動以及相關(guān)機制奠定理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R562

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李琦,錢桂生,張青,王長征;不同劑量脂多糖對大鼠急性肺損傷效應(yīng)的觀察[J];第三軍醫(yī)大學(xué)學(xué)報;2004年10期

2 解翔彬;袁世熒;李進(jìn);姚尚龍;;高張-高膠滲混合液對脂多糖急性肺損傷大鼠的保護(hù)作用[J];國際麻醉學(xué)與復(fù)蘇雜志;2006年02期

3 李琦 ,錢桂生,張青,張倫基,陳仲祥;脂多糖介導(dǎo)的急性肺損傷大鼠血漿IL-13含量的變化[J];解放軍醫(yī)學(xué)雜志;2003年02期

4 雷鳴;倪云峰;李小飛;張志培;劉濤;程慶書;;曲古菌素A對脂多糖致急性肺損傷小鼠的保護(hù)作用觀察[J];解放軍醫(yī)學(xué)雜志;2012年04期

5 馬濤;高玉華;孟盡海;張冬梅;;高滲氯化鈉羥乙基淀粉40注射液對內(nèi)毒素性急性肺損傷大鼠腫瘤壞死因子-α表達(dá)的影響[J];陜西醫(yī)學(xué)雜志;2010年09期

6 鄭翔;季艷梅;郭學(xué)珍;黃云飛;陳黎;;丙酮酸乙酯對LPS致急性肺損傷大鼠肺組織NF-κB p65及TNF-α和IL-lβ的影響[J];微循環(huán)學(xué)雜志;2010年01期

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