低氧和糖皮质激素上调stomatin的机制及其生物学意义
发布时间：2019-03-12 10:58
【摘要】：缺氧是最重要的应激反应之一,是也临床最常见的病理生理过程,与心血管疾病和高原性肺水肿等的发生密切相关。糖皮质激素作为体内最重要的应激激素,在机体对低氧适应中发挥重要作用。人工合成的糖皮质激素-地塞米松(Dex)在临床上被广泛用于治疗肺水肿和肺损伤,它的这种作用是通过降低血管内皮细胞的通透性、加速肺泡腔中液体的清除以及增强肺上皮屏障功能来实现的。 Stomatin是一个重要的脂筏标志蛋白,它广泛表达在人和小鼠的各组织细胞中,参与脂筏结构的装配,囊泡运输、物质的跨膜转运、以及对离子通道和细胞骨架的调节等生理过程。但是迄今对stomatin的功能了解的有限,对其在生理和病理条件下的调节研究的更少。我们前期研究了低氧和地塞米松(Dex)单独及联合作用对大鼠肺脏和人肺腺癌A549细胞stomatin表达的影响。结果发现低氧和Dex在体内和体外均可以上调stomatin的表达。本课题将从细胞和整体水平进一步确定糖皮质激素和低氧单独或共同作用对stomatin基因表达的影响,在此基础上,重点研究低氧和地塞米松上调stomatin表达的机制,并初步探讨stomatin表达上调的生物学意义。 采用Real time-PCR和Western Blot的方法,我们首先在大鼠肺脏和原代培养的肺泡上皮细胞水平进一步证实了地塞米松和低氧不仅单独可诱导stomatin的表达,二者还能联合上调stomatin表达。接下来我们研究了低氧和地塞米松上调stomatin的机制。研究发现,stomatin mRNA有一个相当长的半衰期,约为32小时,低氧和地塞米松处理都能够增强stomatin mRNA的稳定性,使它的半衰期分别延长至原来的1.22倍和1.5倍。我们用荧光素酶报告基因实验研究发现,低氧不能诱导stomatin启动子的转录活性,与低氧不同的是,地塞米松能够以时间和浓度依赖性的方式诱导stomatin启动子的活性,这个结果表明地塞米松也能够直接在转录水平上诱导stomatin mRNA的表达。为了进一步定位stomatin启动子对糖皮质激素诱导反应的位点,我们构建了一系列含stomatin启动子5’端不同截短形式的报告基因载体,荧光素酶活性分析试验表明,启动子的-162至+244区域是对糖皮质激素诱导反应所不可缺少的,而且这一区域在多种细胞类型中均贡献了stomatin基因的绝大部分基础转录活性,因此我们推测这一区域是它的核心启动子区。随后,我们用软件预测了这一区域所包含的可能介导糖皮质激素诱导反应的转录因子结合位点,并且构建了这些位点的相应突变型报告基因载体。结果发现,其中一个位点GRE3,突变之后能够使stomatin启动子失去对地塞米松的诱导反应,这说明GRE3介导了地塞米松对stomatin的诱导反应。因此我们认为,地塞米松在转录水平上对stomatin的诱导是通过激活的糖皮质激素受体与这个GRE3位点结合来实现的。 最后,我们用激光共聚焦显微镜的方法观察了A549细胞中stomatin的表达与actin细胞骨架的关系。结果发现,stomatin在膜周与actin细胞骨架存在共定位。低氧暴露或Dex处理均能使膜周actin增加,干扰内源性stomatin表达后低氧暴露或Dex处理A549细胞都能使actin细胞骨架在膜周分布显著减少,表明低氧和Dex能通过上调stomatin表达,增加与细胞膜连接的细胞骨架,从而增强肺泡上皮细胞膜的稳定性。这可能是低氧和GC增强肺泡上皮细胞屏障的功能,从而使得机体产生对低氧适应性反应的机制之一。
[Abstract]:Hypoxia is one of the most important stress responses, and is also the most common pathophysiological process in the clinic, which is closely related to the occurrence of cardiovascular disease and high altitude pulmonary edema. Glucocorticoid, as the most important stress hormone in the body, plays an important role in the adaptation of hypoxia. The synthetic glucocorticoid-dexamethasone (Dex) is widely used in the treatment of pulmonary edema and lung injury, which is achieved by reducing the permeability of the vascular endothelial cells, accelerating the removal of the liquid in the alveolar cavity, and enhancing the pulmonary epithelial barrier function. Stomatin is an important protein of the lipid raft, which is widely expressed in the tissue cells of human and mouse, is involved in the assembly of the lipid raft structure, the transport of the vesicles, the transport of the substance, and the regulation of the ion channel and the cytoskeleton, and so on. Process. But to date, there is a limited understanding of the function of stomatin, which has been studied under physiological and pathological conditions. Less. We studied the expression of stomatin in lung and human lung adenocarcinoma A549 cells by hypoxia and dexamethasone (Dex) alone and in combination. The results showed that both hypoxia and Dex could be up-regulated in vivo and in vitro. The effect of glucocorticoid and hypoxia on the expression of the statin gene is further determined from the cell and the whole level. On the basis of this, the mechanism of up-regulation of the expression of stomatin by hypoxia and dexamethasone is mainly studied. Meantime, with the method of Real time-PCR and Western Blot, we first confirmed that the expression of dexamethasone and hypoxia not only can induce the expression of the statin in the lung and the primary cultured alveolar epithelial cells of the rat, but can also be used to upregulate the staoma. Tin's expression. Next we studied hypoxia and dexamethasone up-regulation of stomat. In this study, it was found that the stomatin mRNA has a long half-life, which is about 32 hours, and the hypoxia and dexamethasone treatment can enhance the stability of the stomatin mRNA and prolong the half-life of the statin mRNA to the original 1.22-fold. We found that hypoxia could not induce the transcriptional activity of the statostatin promoter, and it was different from the hypoxia that the dexamethasone can induce stomatin in a time-and concentration-dependent manner. The results of the activity of the mover indicate that the dexamethasone can also directly induce the statin mR at the level of transcription. in order to further position the site of the stomatin promoter to the glucocorticoid response, a series of reporter vector, luciferase activity, The analysis shows that the region of-162 to + 244 of the promoter is indispensable to the response of the glucocorticoid, and the region contributes most of the basic transcriptional activity of the stomatin gene in a variety of cell types, so we assume that this region is its nucleus. The core promoter region. Subsequently, we used the software to predict the transcription factor binding site that could mediate the glucocorticoid-mediated response contained in this region, and the corresponding mutant reports of these sites were constructed The results showed that GRE3 in one of the sites, after the mutation, can cause the stomatin promoter to lose the response to dexamethasone, which indicates that the GRE3 mediates the effect of dexamethasone on the stomatin. It is therefore believed that the induction of the statin at the level of transcription by dexamethasone is by activating the glucocorticoid receptor with this GRE3 site. Finally, we used a laser confocal microscope to observe the expression of stomatin in A549 cells and acti. The relationship of the cytoskeleton of n cells was found. The results showed that the statin was fine in the membrane and actin. In the presence of a co-localization of the cytoskeleton, the hypoxia exposure or the Dex treatment can increase the expression of actin cytoskeleton in the membrane around the membrane cycle, and it is shown that the hypoxia and Dex can increase the expression, increase and the cells of the actin cytoskeleton by up-regulation of the statin expression. The membrane is attached to the cytoskeleton to enhance the alveoli. The stability of the skin cell membrane. This may be a function of hypoxia and GC to enhance the barrier of the alveolar epithelial cells, making it possible for the body to adapt to hypoxia
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:Hypoxia is one of the most important stress responses, and is also the most common pathophysiological process in the clinic, which is closely related to the occurrence of cardiovascular disease and high altitude pulmonary edema. Glucocorticoid, as the most important stress hormone in the body, plays an important role in the adaptation of hypoxia. The synthetic glucocorticoid-dexamethasone (Dex) is widely used in the treatment of pulmonary edema and lung injury, which is achieved by reducing the permeability of the vascular endothelial cells, accelerating the removal of the liquid in the alveolar cavity, and enhancing the pulmonary epithelial barrier function. Stomatin is an important protein of the lipid raft, which is widely expressed in the tissue cells of human and mouse, is involved in the assembly of the lipid raft structure, the transport of the vesicles, the transport of the substance, and the regulation of the ion channel and the cytoskeleton, and so on. Process. But to date, there is a limited understanding of the function of stomatin, which has been studied under physiological and pathological conditions. Less. We studied the expression of stomatin in lung and human lung adenocarcinoma A549 cells by hypoxia and dexamethasone (Dex) alone and in combination. The results showed that both hypoxia and Dex could be up-regulated in vivo and in vitro. The effect of glucocorticoid and hypoxia on the expression of the statin gene is further determined from the cell and the whole level. On the basis of this, the mechanism of up-regulation of the expression of stomatin by hypoxia and dexamethasone is mainly studied. Meantime, with the method of Real time-PCR and Western Blot, we first confirmed that the expression of dexamethasone and hypoxia not only can induce the expression of the statin in the lung and the primary cultured alveolar epithelial cells of the rat, but can also be used to upregulate the staoma. Tin's expression. Next we studied hypoxia and dexamethasone up-regulation of stomat. In this study, it was found that the stomatin mRNA has a long half-life, which is about 32 hours, and the hypoxia and dexamethasone treatment can enhance the stability of the stomatin mRNA and prolong the half-life of the statin mRNA to the original 1.22-fold. We found that hypoxia could not induce the transcriptional activity of the statostatin promoter, and it was different from the hypoxia that the dexamethasone can induce stomatin in a time-and concentration-dependent manner. The results of the activity of the mover indicate that the dexamethasone can also directly induce the statin mR at the level of transcription. in order to further position the site of the stomatin promoter to the glucocorticoid response, a series of reporter vector, luciferase activity, The analysis shows that the region of-162 to + 244 of the promoter is indispensable to the response of the glucocorticoid, and the region contributes most of the basic transcriptional activity of the stomatin gene in a variety of cell types, so we assume that this region is its nucleus. The core promoter region. Subsequently, we used the software to predict the transcription factor binding site that could mediate the glucocorticoid-mediated response contained in this region, and the corresponding mutant reports of these sites were constructed The results showed that GRE3 in one of the sites, after the mutation, can cause the stomatin promoter to lose the response to dexamethasone, which indicates that the GRE3 mediates the effect of dexamethasone on the stomatin. It is therefore believed that the induction of the statin at the level of transcription by dexamethasone is by activating the glucocorticoid receptor with this GRE3 site. Finally, we used a laser confocal microscope to observe the expression of stomatin in A549 cells and acti. The relationship of the cytoskeleton of n cells was found. The results showed that the statin was fine in the membrane and actin. In the presence of a co-localization of the cytoskeleton, the hypoxia exposure or the Dex treatment can increase the expression of actin cytoskeleton in the membrane around the membrane cycle, and it is shown that the hypoxia and Dex can increase the expression, increase and the cells of the actin cytoskeleton by up-regulation of the statin expression. The membrane is attached to the cytoskeleton to enhance the alveoli. The stability of the skin cell membrane. This may be a function of hypoxia and GC to enhance the barrier of the alveolar epithelial cells, making it possible for the body to adapt to hypoxia
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【共引文献】
相关期刊论文 前5条
1 王雁;崔宇辉;张金涛;;Stomatin在哺乳动物机械感觉传导中的作用[J];东南国防医药;2010年02期
2 常栋;王天佑;刘芝华;;stomatin家族与恶性肿瘤[J];中华临床医师杂志(电子版);2011年02期
3 田启宇;陈永辉;范志勇;李铁军;贺建华;邓近平;;炎热气候条件下母猪妊娠后期不同组织中GRα mRNA的表达规律研究[J];中国农学通报;2013年29期
4 林雪彩;孙红英;;GR亚型α和β在口腔扁平苔藓中的表达及临床分析[J];中华临床医师杂志(电子版);2013年12期
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