LPS诱导下A549细胞的TMEM16A表达及功能
发布时间：2018-01-07 05:13
本文关键词：LPS诱导下A549细胞的TMEM16A表达及功能 出处：《河北医科大学》2012年硕士论文 论文类型：学位论文
更多相关文章： ALI ALI/ARDS LPS TMEM16A TNF-α
【摘要】：目的：急性肺损伤（acute lung injury,ALI）是机体遭受各种诱因严重打击引起的以肺泡毛细血管膜通透性增加为特征的肺部炎症综合症。发病急、病情重，未经有效治疗可以发展为急性呼吸窘迫综合症(acuterespiratory distress syndrome，ARDS)。ALI病因与发病机理复杂，而肺间质与肺泡水肿却是共同病理表现，说明ALI/ARDS均存在肺水清除障碍。 既往有关ALI/ARDS研究主要集中在肺内炎症过程，近年来对ALI肺水形成与清除过程给予较多关注，因此与水转运有关的离子通道研究逐渐活跃。TMEM16A就是TMEM家族中近来受到关注的与细胞分泌相关的新型成员。已初步发现其在大鼠ALI模型肺泡上皮细胞中表达明显增强，且不同诱因所致ALI大鼠模型之间还有差异。因此，有必要进一步探讨该蛋白活性与ALI/ARDS肺水清除的关系。 TMEM16A基因,又名TAOS2, DOG1, FLJ10261或ORAOV2，是TMEM家族的一员,已有研究证明在食管癌、膀胱癌和乳腺癌中，感觉神经元，上皮细胞均有表达。于2008年CaputoA等证实该蛋白为钙激活的氯离子通道，有研究表明其与气道的粘液腺体分泌相关。目前认为Ⅱ型上皮细胞（Alveolar epithelial cells typeⅡ，ATⅡ）在肺液清除中起着关键作用，一些研究正在探索肺泡Ⅱ型上皮细胞上氯离子通道的特点。Yang C近期一项研究提示：Ⅱ型上皮细胞参与了与尿嘌呤三磷酸腺苷（UTP）相关的液体分泌，Rock JR等研究表明：TMEM16A也参与了与UTP相关的液体分泌。而TMEM16A作为一种氯离子通道，其在急性肺损伤肺水清除中的作用尚无研究。 感染是引起肺损伤和呼吸功能障碍的常见原因，脂多糖（LPS）是细菌内毒素的主要成分,可刺激组织细胞释放多种炎性物质（包括细胞因子）、诱导炎症反应。临床资料表明严重感染与ALI/ARDS均有密切关系，但应用不同浓度的LPS刺激肺泡上皮细胞TMEM16A的表达，国内外尚未见明确报道。因此本实验的目的即： 1.检测脂多糖(LPS)刺激下A549细胞上清液中TNF-α的浓度，了解其与细胞损伤程度的关系。 2.检测A549肺泡上皮细胞上是否有TMEM16A蛋白的阳性表达，以及观察不同浓度的脂多糖(LPS)诱导下以A549细胞TMEM16A蛋白表达水平的差异为进一步探讨该蛋白活性与ALI/ARDS肺水清除的关系提供新的思路和实验依据。 方法： 1.A549细胞分为对照组和实验组，实验组分别用不同终浓度的LPS(5μg/ml；10μg/ml；15μg/ml)处理24h~[1-3]； 2. MTT法检测LPS对A549细胞增殖的影响，电镜观察细胞器的变化； 3.采用ELISA法检测脂多糖(LPS)刺激下A549细胞上清液中TNF-α的浓度； 4. Western blot法检测对照组和实验组A549细胞TMEM16A蛋白表达水平。 结果: 1.细胞形态学及细胞器变化的结果 倒置显微镜结果：对照组：A549细胞为多角形、椭圆形，胞质非常丰富，细胞突起比较明显，呈贴壁性生长，并融合成片，基本上无核固缩；实验组脂多糖处理后(5μg/ml)，A549细胞的形态变化与对照组无明显变化。脂多糖处理后(10μg/ml和15μg/ml),A549细胞形态变圆，体积缩小，胞质内可出现大小不等的颗粒。电镜结果：对照组A549细胞表面有大量的短小微绒毛，细胞质丰富，，内有各种细胞器如线粒体、溶酶体、内质网、高尔基复合体、板层小体、粘液空泡和微囊等，实验组脂多糖处理后(5μg/ml)，A549细胞的细胞器变化与对照组无明显变化。脂多糖(10μg/ml和15μg/ml)处理后,A549细胞表面的微绒毛减少，细胞内可见大量的空泡，线粒体部分嵴和少部分膜融合，模糊不清，可见嵴的缺失现象。粗面内质网轻度扩张，可见颗粒融合及脱颗粒现象。且随着脂多糖浓度的增加细胞器损伤逐渐加重。 2．MTT法检测不同时间不同浓度脂多糖干预的细胞生长抑制率较对照组逐渐增加，A549细胞的生长抑制率实验组5μg/ml，10μg/ml，15μg/ml的细胞生长抑制率逐渐增加，5μg/ml实验组在脂多糖刺激3小时与6小时后细胞生长抑制率比较不具有统计学意义，其余实验组在任何时间点组间两两比较均具有统计学意义(p0.01)。而在同一时间点，3，12，24小时10μg/ml和15μg/ml实验组间比较均不具有统计学意义(p0.05)，其余实验组在同一时间点组间两两比较均具有统计学意义(p0.01)。 3.ELISA法检测结果显示实验组细胞培养上清液TNF-α浓度较对照组均明显增加，而且差异均具有统计学意义(p0.01)。 4.Western-blot实验各组TMEM16A的表达：实验组5μg/ml（0.3028±0.01898），10μg/ml（0.2946±0.00535）的TMEM16A的蛋白表达量较对照组（0.2764±0.00814）升高，15μg/ml（0.2190±0.00865）组细胞的TMEM16A的蛋白表达量较对照组降低，5μg/ml的实验组升高与对照组总体均数相比具有统计学意义（p=0.014），15μg/ml蛋白表达水平降低比对照组及其他实验组总体均数相比均具有统计学意义(p0.001)。 结论: 1.脂多糖可以成功诱导A549细胞的急性损伤； 2.对照组及实验组细胞培养上清液中TNF-α水平在不同浓度，不同时间脂多糖干预下的TNF-α的水平随着细胞损害程度的加重呈逐渐升高趋势，各实验组组间变化具有统计学差异； 3.Western blot法证明A549细胞上有TMEM16A的蛋白阳性表达。细胞损伤较轻，TMEM16A的蛋白表达水平应激性升高，但在细胞结构遭受严重损伤时，细胞形态结构及功能均已损坏，TMEM16A的蛋白表达水平明显降低。
[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.
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