發(fā)布時間：2018-01-07 05:13

  本文關(guān)鍵詞：LPS誘導下A549細胞的TMEM16A表達及功能　出處：《河北醫(yī)科大學》2012年碩士論文　論文類型：學位論文

  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）是機體遭受各種誘因嚴重打擊引起的以肺泡毛細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)病機理復雜，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ALI肺水形成與清除過程給予較多關(guān)注，因此與水轉(zhuǎn)運有關(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模型肺泡上皮細胞中表達明顯增強，且不同誘因所致ALI大鼠模型之間還有差異。因此，有必要進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，上皮細胞均有表達。于2008年CaputoA等證實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)。目前認為Ⅱ型上皮細胞（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型上皮細胞上氯離子通道的特點。Yang C近期一項研究提示：Ⅱ型上皮細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）是細菌內(nèi)毒素的主要成分,可刺激組織細胞釋放多種炎性物質(zhì)（包括細胞因子）、誘導炎癥反應。臨床資料表明嚴重感染與ALI/ARDS均有密切關(guān)系，但應用不同濃度的LPS刺激肺泡上皮細胞TMEM16A的表達，國內(nèi)外尚未見明確報道。因此本實驗的目的即： 1.檢測脂多糖(LPS)刺激下A549細胞上清液中TNF-α的濃度，了解其與細胞損傷程度的關(guān)系。 2.檢測A549肺泡上皮細胞上是否有TMEM16A蛋白的陽性表達，以及觀察不同濃度的脂多糖(LPS)誘導下以A549細胞TMEM16A蛋白表達水平的差異為進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驗依據(jù)。 方法： 1.A549細胞分為對照組和實驗組，實驗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A549細胞增殖的影響，電鏡觀察細胞器的變化； 3.采用ELISA法檢測脂多糖(LPS)刺激下A549細胞上清液中TNF-α的濃度； 4. Western blot法檢測對照組和實驗組A549細胞TMEM16A蛋白表達水平。 結(jié)果: 1.細胞形態(tài)學及細胞器變化的結(jié)果 倒置顯微鏡結(jié)果：對照組：A549細胞為多角形、橢圓形，胞質(zhì)非常豐富，細胞突起比較明顯，呈貼壁性生長，并融合成片，基本上無核固縮；實驗組脂多糖處理后(5μg/ml)，A549細胞的形態(tài)變化與對照組無明顯變化。脂多糖處理后(10μg/ml和15μg/ml),A549細胞形態(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：對照組A549細胞表面有大量的短小微絨毛，細胞質(zhì)豐富，，內(nèi)有各種細胞器如線粒體、溶酶體、內(nèi)質(zhì)網(wǎng)、高爾基復合體、板層小體、粘液空泡和微囊等，實驗組脂多糖處理后(5μg/ml)，A549細胞的細胞器變化與對照組無明顯變化。脂多糖(10μg/ml和15μg/ml)處理后,A549細胞表面的微絨毛減少，細胞內(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胞器損傷逐漸加重。 2．MTT法檢測不同時間不同濃度脂多糖干預的細胞生長抑制率較對照組逐漸增加，A549細胞的生長抑制率實驗組5μg/ml，10μg/ml，15μg/ml的細胞生長抑制率逐漸增加，5μg/ml實驗組在脂多糖刺激3小時與6小時后細胞生長抑制率比較不具有統(tǒng)計學意義，其余實驗組在任何時間點組間兩兩比較均具有統(tǒng)計學意義(p0.01)。而在同一時間點，3，12，24小時10μg/ml和15μg/ml實驗組間比較均不具有統(tǒng)計學意義(p0.05)，其余實驗組在同一時間點組間兩兩比較均具有統(tǒng)計學意義(p0.01)。 3.ELISA法檢測結(jié)果顯示實驗組細胞培養(yǎng)上清液TNF-α濃度較對照組均明顯增加，而且差異均具有統(tǒng)計學意義(p0.01)。 4.Western-blot實驗各組TMEM16A的表達：實驗組5μg/ml（0.3028±0.01898），10μg/ml（0.2946±0.00535）的TMEM16A的蛋白表達量較對照組（0.2764±0.00814）升高，15μg/ml（0.2190±0.00865）組細胞的TMEM16A的蛋白表達量較對照組降低，5μg/ml的實驗組升高與對照組總體均數(shù)相比具有統(tǒng)計學意義（p=0.014），15μg/ml蛋白表達水平降低比對照組及其他實驗組總體均數(shù)相比均具有統(tǒng)計學意義(p0.001)。 結(jié)論: 1.脂多糖可以成功誘導A549細胞的急性損傷； 2.對照組及實驗組細胞培養(yǎng)上清液中TNF-α水平在不同濃度，不同時間脂多糖干預下的TNF-α的水平隨著細胞損害程度的加重呈逐漸升高趨勢，各實驗組組間變化具有統(tǒng)計學差異； 3.Western blot法證明A549細胞上有TMEM16A的蛋白陽性表達。細胞損傷較輕，TMEM16A的蛋白表達水平應激性升高，但在細胞結(jié)構(gòu)遭受嚴重損傷時，細胞形態(tài)結(jié)構(gòu)及功能均已損壞，TMEM16A的蛋白表達水平明顯降低。
[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

本文編號：1391117