發(fā)布時(shí)間：2018-01-07 05:13

  本文關(guān)鍵詞：LPS誘導(dǎo)下A549細(xì)胞的TMEM16A表達(dá)及功能　出處：《河北醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： ALI ALI/ARDS LPS TMEM16A TNF-α

【摘要】：目的：急性肺損傷（acute lung injury,ALI）是機(jī)體遭受各種誘因嚴(yán)重打擊引起的以肺泡毛細(xì)血管膜通透性增加為特征的肺部炎癥綜合癥。發(fā)病急、病情重，未經(jīng)有效治療可以發(fā)展為急性呼吸窘迫綜合癥(acuterespiratory distress syndrome，ARDS)。ALI病因與發(fā)病機(jī)理復(fù)雜，而肺間質(zhì)與肺泡水腫卻是共同病理表現(xiàn)，說明ALI/ARDS均存在肺水清除障礙。 既往有關(guān)ALI/ARDS研究主要集中在肺內(nèi)炎癥過程，近年來對(duì)ALI肺水形成與清除過程給予較多關(guān)注，因此與水轉(zhuǎn)運(yùn)有關(guān)的離子通道研究逐漸活躍。TMEM16A就是TMEM家族中近來受到關(guān)注的與細(xì)胞分泌相關(guān)的新型成員。已初步發(fā)現(xiàn)其在大鼠ALI模型肺泡上皮細(xì)胞中表達(dá)明顯增強(qiáng)，且不同誘因所致ALI大鼠模型之間還有差異。因此，有必要進(jìn)一步探討該蛋白活性與ALI/ARDS肺水清除的關(guān)系。 TMEM16A基因,又名TAOS2, DOG1, FLJ10261或ORAOV2，是TMEM家族的一員,已有研究證明在食管癌、膀胱癌和乳腺癌中，感覺神經(jīng)元，上皮細(xì)胞均有表達(dá)。于2008年CaputoA等證實(shí)該蛋白為鈣激活的氯離子通道，有研究表明其與氣道的粘液腺體分泌相關(guān)。目前認(rèn)為Ⅱ型上皮細(xì)胞（Alveolar epithelial cells typeⅡ，ATⅡ）在肺液清除中起著關(guān)鍵作用，一些研究正在探索肺泡Ⅱ型上皮細(xì)胞上氯離子通道的特點(diǎn)。Yang C近期一項(xiàng)研究提示：Ⅱ型上皮細(xì)胞參與了與尿嘌呤三磷酸腺苷（UTP）相關(guān)的液體分泌，Rock JR等研究表明：TMEM16A也參與了與UTP相關(guān)的液體分泌。而TMEM16A作為一種氯離子通道，其在急性肺損傷肺水清除中的作用尚無研究。 感染是引起肺損傷和呼吸功能障礙的常見原因，脂多糖（LPS）是細(xì)菌內(nèi)毒素的主要成分,可刺激組織細(xì)胞釋放多種炎性物質(zhì)（包括細(xì)胞因子）、誘導(dǎo)炎癥反應(yīng)。臨床資料表明嚴(yán)重感染與ALI/ARDS均有密切關(guān)系，但應(yīng)用不同濃度的LPS刺激肺泡上皮細(xì)胞TMEM16A的表達(dá)，國內(nèi)外尚未見明確報(bào)道。因此本實(shí)驗(yàn)的目的即： 1.檢測脂多糖(LPS)刺激下A549細(xì)胞上清液中TNF-α的濃度，了解其與細(xì)胞損傷程度的關(guān)系。 2.檢測A549肺泡上皮細(xì)胞上是否有TMEM16A蛋白的陽性表達(dá)，以及觀察不同濃度的脂多糖(LPS)誘導(dǎo)下以A549細(xì)胞TMEM16A蛋白表達(dá)水平的差異為進(jìn)一步探討該蛋白活性與ALI/ARDS肺水清除的關(guān)系提供新的思路和實(shí)驗(yàn)依據(jù)。 方法： 1.A549細(xì)胞分為對(duì)照組和實(shí)驗(yàn)組，實(shí)驗(yàn)組分別用不同終濃度的LPS(5μg/ml；10μg/ml；15μg/ml)處理24h~[1-3]； 2. MTT法檢測LPS對(duì)A549細(xì)胞增殖的影響，電鏡觀察細(xì)胞器的變化； 3.采用ELISA法檢測脂多糖(LPS)刺激下A549細(xì)胞上清液中TNF-α的濃度； 4. Western blot法檢測對(duì)照組和實(shí)驗(yàn)組A549細(xì)胞TMEM16A蛋白表達(dá)水平。 結(jié)果: 1.細(xì)胞形態(tài)學(xué)及細(xì)胞器變化的結(jié)果 倒置顯微鏡結(jié)果：對(duì)照組：A549細(xì)胞為多角形、橢圓形，胞質(zhì)非常豐富，細(xì)胞突起比較明顯，呈貼壁性生長，并融合成片，基本上無核固縮；實(shí)驗(yàn)組脂多糖處理后(5μg/ml)，A549細(xì)胞的形態(tài)變化與對(duì)照組無明顯變化。脂多糖處理后(10μg/ml和15μg/ml),A549細(xì)胞形態(tài)變圓，體積縮小，胞質(zhì)內(nèi)可出現(xiàn)大小不等的顆粒。電鏡結(jié)果：對(duì)照組A549細(xì)胞表面有大量的短小微絨毛，細(xì)胞質(zhì)豐富，，內(nèi)有各種細(xì)胞器如線粒體、溶酶體、內(nèi)質(zhì)網(wǎng)、高爾基復(fù)合體、板層小體、粘液空泡和微囊等，實(shí)驗(yàn)組脂多糖處理后(5μg/ml)，A549細(xì)胞的細(xì)胞器變化與對(duì)照組無明顯變化。脂多糖(10μg/ml和15μg/ml)處理后,A549細(xì)胞表面的微絨毛減少，細(xì)胞內(nèi)可見大量的空泡，線粒體部分嵴和少部分膜融合，模糊不清，可見嵴的缺失現(xiàn)象。粗面內(nèi)質(zhì)網(wǎng)輕度擴(kuò)張，可見顆粒融合及脫顆�，F(xiàn)象。且隨著脂多糖濃度的增加細(xì)胞器損傷逐漸加重。 2．MTT法檢測不同時(shí)間不同濃度脂多糖干預(yù)的細(xì)胞生長抑制率較對(duì)照組逐漸增加，A549細(xì)胞的生長抑制率實(shí)驗(yàn)組5μg/ml，10μg/ml，15μg/ml的細(xì)胞生長抑制率逐漸增加，5μg/ml實(shí)驗(yàn)組在脂多糖刺激3小時(shí)與6小時(shí)后細(xì)胞生長抑制率比較不具有統(tǒng)計(jì)學(xué)意義，其余實(shí)驗(yàn)組在任何時(shí)間點(diǎn)組間兩兩比較均具有統(tǒng)計(jì)學(xué)意義(p0.01)。而在同一時(shí)間點(diǎn)，3，12，24小時(shí)10μg/ml和15μg/ml實(shí)驗(yàn)組間比較均不具有統(tǒng)計(jì)學(xué)意義(p0.05)，其余實(shí)驗(yàn)組在同一時(shí)間點(diǎn)組間兩兩比較均具有統(tǒng)計(jì)學(xué)意義(p0.01)。 3.ELISA法檢測結(jié)果顯示實(shí)驗(yàn)組細(xì)胞培養(yǎng)上清液TNF-α濃度較對(duì)照組均明顯增加，而且差異均具有統(tǒng)計(jì)學(xué)意義(p0.01)。 4.Western-blot實(shí)驗(yàn)各組TMEM16A的表達(dá)：實(shí)驗(yàn)組5μg/ml（0.3028±0.01898），10μg/ml（0.2946±0.00535）的TMEM16A的蛋白表達(dá)量較對(duì)照組（0.2764±0.00814）升高，15μg/ml（0.2190±0.00865）組細(xì)胞的TMEM16A的蛋白表達(dá)量較對(duì)照組降低，5μg/ml的實(shí)驗(yàn)組升高與對(duì)照組總體均數(shù)相比具有統(tǒng)計(jì)學(xué)意義（p=0.014），15μg/ml蛋白表達(dá)水平降低比對(duì)照組及其他實(shí)驗(yàn)組總體均數(shù)相比均具有統(tǒng)計(jì)學(xué)意義(p0.001)。 結(jié)論: 1.脂多糖可以成功誘導(dǎo)A549細(xì)胞的急性損傷； 2.對(duì)照組及實(shí)驗(yàn)組細(xì)胞培養(yǎng)上清液中TNF-α水平在不同濃度，不同時(shí)間脂多糖干預(yù)下的TNF-α的水平隨著細(xì)胞損害程度的加重呈逐漸升高趨勢，各實(shí)驗(yàn)組組間變化具有統(tǒng)計(jì)學(xué)差異； 3.Western blot法證明A549細(xì)胞上有TMEM16A的蛋白陽性表達(dá)。細(xì)胞損傷較輕，TMEM16A的蛋白表達(dá)水平應(yīng)激性升高，但在細(xì)胞結(jié)構(gòu)遭受嚴(yán)重?fù)p傷時(shí)，細(xì)胞形態(tài)結(jié)構(gòu)及功能均已損壞，TMEM16A的蛋白表達(dá)水平明顯降低。
[Abstract]:Objective: acute lung injury (acute lung, injury, ALI) is a serious blow to the body caused by various incentives to suffer the increased permeability of alveolar capillary pulmonary inflammation syndrome characterized by acute onset, severe illness, acute respiratory distress syndrome development without effective treatment can (acuterespiratory distress syndrome, ARDS) the etiology and pathogenesis of.ALI complex, pulmonary interstitial and alveolar edema are common pathological manifestations, the lung water to remove barriers are ALI/ARDS.
Previous studies of ALI/ARDS mainly focused on the inflammatory process, in recent years, the ALI lung water formation and clearance given more attention, more attention so the research related to water transport ion channel.TMEM16A is gradually active in the TMEM family members related to secretion of new cells have been discovered. The enhanced expression of ALI in rats the model of alveolar epithelial cells, and ALI rat model induced by different causes and differences. Therefore, it is necessary to further explore the relationship between the activity of ALI/ARDS protein and lung water clearance.
TMEM16A gene, also known as TAOS2, DOG1, FLJ10261 or ORAOV2, is a member of the TMEM family, it has been shown that the sensory neurons in esophageal cancer, bladder cancer and breast cancer, and is expressed in epithelial cells. In 2008, CaputoA confirmed that the protein is a calcium chloride ion channel activation, studies have shown that the airway mucus the glandular secretion. Type II epithelial cells (Alveolar epithelial cells type II, AT II) in lung plays a key role in fluid clearance, some research is to explore the characteristics of type II alveolar epithelial cells on the chloride channel.Yang C a recent study suggests that type II epithelial cells involved in ATP and purine (UTP) related to the liquid secretion, Rock JR shows that the TMEM16A is involved in UTP related fluid secretion. TMEM16A is a chloride channel, its role in acute lung injury of lung water clearance in Shang No research.
Infection is a common cause of lung damage and respiratory dysfunction, lipopolysaccharide (LPS) is a major component of bacterial endotoxin, which may stimulate the cells release a variety of inflammatory substances (including cytokines), induced inflammation. Clinical data suggest that severe infection and ALI/ARDS were closely related, but the application of different concentrations of LPS to stimulate the expression of alveolar epithelial cells TMEM16A, at home and abroad has not been reported yet. Therefore, the objective of this experiment is:
1. to detect the concentration of TNF- alpha in the supernatant of A549 cells stimulated by lipopolysaccharide (LPS), and to understand the relationship between the level of cell injury and the degree of cell injury.
If there is a positive expression of TMEM16A 2. was detected by A549 in alveolar epithelial cells, and the effects of different concentration of lipopolysaccharide (LPS) expression in A549 cells induced by TMEM16A protein in order to provide ideas and new experimental evidence to further explore the relationship between the activity of ALI/ARDS protein and lung water clearance.
Method錛

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