本文選題：機械牽張 + 張力敏感性陽離子通道��； 參考：《重慶醫(yī)科大學》2011年博士論文

【摘要】：目的分別觀察呼吸機機械通氣、機械牽張對兔氣道黏膜和人氣道黏膜上皮細胞(HBE16)黏蛋白(MUC)5AC表達的影響,并對其信號通路機制進行初步探討。 方法日本大耳兔隨機分為對照組、機械通氣1、3、6、12和24h組以及2、5和10cmH2O呼氣末正壓(PEEP)組。取支氣管肺泡灌洗液(BALF),用ELISA和RT-PCR法檢測BALF中腫瘤壞死因子(TNF)-α、白介素(IL)-8、MUC5AC蛋白和mRNA的水平,計數(shù)BALF中多型核粒細胞,底物檢測法測定中性粒細胞彈力酶(NE)活性。人氣道黏膜上皮細胞(HBE16)體外培養(yǎng),采用小型生物撞擊機給予機械牽張刺激,各組培養(yǎng)細胞依施加條件不同而分為對照、牽張、牽張+釓、牽張+硝苯吡啶、牽張+低分子量肝素、牽張+ MARCKS效應結構域(ED)鎖核酸(LNA)以及牽張+ MARCKS的ED無關對照LNA序列共7組,采用流式細胞儀檢測牽張前后胞內(nèi)游離Ca~(2+)熒光強度的變化,激光共聚焦顯微鏡觀察胞內(nèi)游離Ca~(2+)分布的情況,并分別采用逆轉(zhuǎn)錄聚合酶鏈反應(RT-PCR)和免疫熒光法觀察與胞吐相關的突觸相關膜蛋白SNAP23以及黏蛋白(MUC)5AC mRNA和蛋白表達,酶聯(lián)免疫吸附試驗(ELISA)方法檢測細胞培養(yǎng)上清中MUC5AC分泌。 再將構建的人氣道黏膜上皮細胞牽張模型,分別給予絲裂素活化蛋白激酶(MAPK)信號通路的三條主要通路抑制劑:JNK抑制劑(SP600125)、p38蛋白激酶抑制劑(SB203580)及ERK1/2抑制劑(U0126)(濃度均為30μmol/L),并設未進行機械牽張的對照組和單純機械牽張組。分別采用RT-PCR和ELISA方法檢測MUC5AC mRNA表達和蛋白分泌量。 結果機械通氣能顯著增強MUC5AC的分泌(P0.05)。通氣3h TNF-α表達至峰值,隨后逐漸下降(P0.05)。通氣6h IL-8表達至峰值,隨后逐漸下降(P0.05)。通氣12h后多型核粒細胞數(shù)及中性粒細胞彈力酶活性顯著升高,24h后回降(P0.05)。隨著PEEP的增加,TNF-α、IL-8、MUC5ACmRNA的表達和蛋白含量、多型核粒細胞數(shù)和NE活性均隨之升高(P0.05)。 機械牽張能顯著升高人氣道黏膜上皮細胞胞內(nèi)Ca~(2+)濃度(P0.01);同時能顯著升高人氣道黏膜上皮細胞中SNAP23和MUC5AC表達,顯著提升細胞培養(yǎng)上清中MUC5AC分泌(P0.05);釓、硝苯吡啶、MARCKS的ED-LNA均能抑制機械牽張對SNAP23表達和MUC5AC表達、分泌的提升作用(均P0.05);而低分子量肝素未能顯著降低SNAP23表達和MUC5AC表達、分泌(P0.05)。U0126和SB203580能抑制牽張所致MUC5AC mRNA和蛋白表達增加(均P0.01)。 結論機械通氣和牽張能促進氣道黏膜上皮細胞MUC5AC mRNA的表達和MUC5AC的分泌,其機制可能與機械通氣和牽張引發(fā)的炎癥反應、張力敏感性陽離子通道、Ca~(2+)內(nèi)流、MARCKS途徑以及ERK1/2、p38 MAPK信號通路有關。
[Abstract]:Objective to observe the effects of ventilator mechanical ventilation and mechanical stretch on the expression of mucin MUC5AC in rabbit airway mucosal epithelial cells (HBE16) and to explore its signal pathway mechanism. Methods Japanese large ear rabbits were randomly divided into two groups: control group, mechanical ventilation group of 12 and 24 hours after mechanical ventilation, and group of 2 and 10cmH2O positive end expiratory pressure (PEEP). Bronchoalveolar lavage fluid (BALF) was used to detect the levels of tumor necrosis factor TNF- 偽 (TNF- 偽), interleukin (IL-8) MUC5AC protein and mRNA in BALF by ELISA and RT-PCR methods. The polymorphonuclear cells in BALF were counted and the activity of neutrophil elastase was determined by substrate assay. In vitro culture of human mucosal epithelial cells (HBE16) was performed with a small biological impact machine. The cultured cells in each group were divided into two groups according to different applied conditions: distraction gadolinium and nifedipine. There were 7 groups of low molecular weight heparin, distraction of MARCKS effect domain EDN) and Ed unrelated control LNA sequence of distraction MARCKS. Flow cytometry was used to detect the fluorescence intensity of intracellular free Ca~(2 before and after distraction. Laser confocal microscopy was used to observe the distribution of intracellular free Ca~(2. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the expression of synaptic associated membrane protein SNAP23 and mucin MUC5AC mRNA and protein, respectively. Enzyme linked immunosorbent assay (Elisa) was used to detect the secretion of MUC5AC in the supernatant of cell culture. Then the human airway epithelial cell stretch model was constructed. The mitogen-activated protein kinase (MAPK) signaling pathway was treated with three major pathway inhibitors: 1 / JNK inhibitor SP600125 / p38 protein kinase inhibitor / SB203580) and ERK1/2 inhibitor (30 渭 mol / L) respectively. The control group without mechanical stretch and the control group with mechanical distraction alone were set up. MUC5AC mRNA expression and protein secretion were detected by RT-PCR and ELISA. Results Mechanical ventilation could significantly enhance the secretion of MUC5AC (P 0.05). The expression of TNF- 偽 reached its peak at 3 h after ventilation, and then decreased gradually. The expression of IL-8 reached its peak at 6 h after ventilation, and then decreased gradually (P 0.05). After 12 hours of ventilation, the number of polymorphonuclear cells and the activity of neutrophil elastase increased significantly after 24 h ventilation. With the increase of PEEP, the expression of MUC5AC mRNA, the number of polymorphonuclear granulocytes and the activity of NE increased with the increase of TNF- 偽 IL-8 mRNA and protein content. Mechanical distraction could significantly increase the concentration of Ca~(2 in the epithelial cells of the human duct, increase the expression of SNAP23 and MUC5AC in the epithelial cells of the human duct, and increase the secretion of MUC5AC in the supernatant of the cell culture. The ED-LNA of nifedipine rcks could inhibit the expression of SNAP23 and MUC5AC and increase the secretion of MUC5AC by mechanical stretch (all P0.05%), but low molecular weight heparin could not significantly decrease the expression of SNAP23 and MUC5AC. The secretion of P0.05, U0126 and SB203580 inhibited the increase of MUC5AC mRNA and protein expression induced by distraction (both P0.01a). Conclusion Mechanical ventilation and stretch can promote the expression of MUC5AC mRNA and the secretion of MUC5AC in airway epithelial cells, which may be related to the inflammation induced by mechanical ventilation and stretch. Tension-sensitive cationic channel (Cam 2) is related to the MARCKS pathway and ERK1 / 2 p38 MAPK signaling pathway.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363.1

【參考文獻】

相關期刊論文 前10條

1 吳小玲;周向東;;氣道黏液高分泌的信號傳導通路[J];基礎醫(yī)學與臨床;2008年11期

2 李琪;周向東;;TNF-α與香煙煙霧在誘導氣道上皮細胞株黏蛋白表達的相互作用[J];基礎醫(yī)學與臨床;2009年11期

3 雷蕊霞;周向東;;血紅素加氧酶-1抑制香煙煙霧提取物致體外人肺A549細胞黏液高分泌[J];基礎醫(yī)學與臨床;2010年03期

4 鄔海橋;丁陽平;周向東;;紅茶提取物在炎性氣道黏液高分泌中的作用[J];上海交通大學學報(醫(yī)學版);2009年02期

5 余紅梅;周向東;;透明質(zhì)烷和CD44參與調(diào)節(jié)氣道黏液高分泌的分子機制[J];上海交通大學學報(醫(yī)學版);2010年01期

6 丁寧;肖慧;高巨;許立新;佘守章;;P38及上游激酶MKK6對周期性牽張應變誘導的肺泡巨噬細胞高遷移率族蛋白B1表達的調(diào)控[J];生理學報;2009年01期

7 姜勇,韓家淮;p38MAPK信號傳導通路[J];生命科學;1999年03期

8 董愛萍;姚銳;;心臟機械牽張激活的信號轉(zhuǎn)導途徑研究進展[J];咸寧學院學報(醫(yī)學版);2008年03期

9 趙芳;程龍獻;曾秋棠;鄔堂春;張濤;史鈺芳;;p38MAPK在機械牽張致大鼠心臟TREK-1通道表達改變中的作用[J];中國病理生理雜志;2008年09期

10 李琪;周向東;;中性粒細胞彈力蛋白酶引起黏蛋白5AC高表達的信號轉(zhuǎn)導機制[J];中華醫(yī)學雜志;2007年05期

，

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