肾上腺素α1受体在人食管下括约肌的表达研究
发布时间：2018-08-16 12:15
【摘要】：目的：原发性食管运动功能障碍性疾病如贲门失弛缓症、胡桃夹食管、高张性LES等是一类严重影响人们生活质量的疾病。这类疾病往往伴有一些典型症状，如下咽困难等。近些年来，随着人群生活水平提高和食管测压和24小时PH检测等检查仪器的应用，原发性食管运动功能障碍性疾病的发现病例有所升高。但有关此类疾病的病因和发病机制尚不完全明确。研究显示，原发性食管运动功能障碍性疾病和多种因素有关。神经、激素调节、精神心理因素、炎症反应、病毒感染及自身免疫因素等均可能参与了原发性食管运动功能障碍性疾病的发病。 人食管下括约肌(Lower esophageal sphincter, LES)位于食管胃连接部。研究显示，LES由套索纤维（sling fibers，SF）和钩状纤维(clasp fibres，CF)组成。其在机体多种因素的调节下协同膈脚调节食管下段腔内压。这个位于食管胃连接部的高压带具有重要的生理意义。如同阀门样的作用使其在生理抗反流中发挥关键作用。而其功能的紊乱可导致多种食管运动功能障碍性疾病。研究此处的调节机制对胃食管返流类疾病和食管运动功能障碍性疾病具有重要意义。 肾上腺素α1受体（α1-AR）及其亚型属于G蛋白偶联受体，在人和哺乳动物体内广泛表达。现有研究提示，α1-AR对于平滑肌收缩的调节较为重要，但既往关于α1-AR表达的研究主要集中于血管、生殖泌尿、肝脏和肾脏等器官。对于胃肠道研究较少。尚未有对人LES的α1-AR受体及其亚型表达分布和功能的研究。 本实验通过蛋白印迹法（Western-blot）和逆转录聚合酶链反应(RT-PCR)来研究α1-AR及其受体亚型α1A-AR, α1B-AR, α1D-AR的mRNA和蛋白受体在食管平滑肌、套索纤维、钩状纤维和胃底平滑肌的表达水平。通过本项研究完善LES受体的表达规律，为此后α1-AR受体功能的研究和治疗食管运动功能障碍性疾病提供理论基础。 方法：选取因高位食管癌行食管大部切除术的患者30例。在手术室收集新鲜食管胃结合部标本，仔细游离套索纤维肌条、钩状纤维肌条、胃底和食管环形肌肌条。提取组织总RNA后反转录为cDNA，应用3种α1-AR的引物行RT-PCR。分析扩增产物中目的条带的光密度值(IOD)。各肌条中mRNA的相对含量为α1-AR各亚型与内参（β-actin）的比值大小代表。提取组织总蛋白，BCA法测量蛋白浓度后将蛋白调整至相同浓度，电泳分离出α1-AR个亚型后转膜，再分别应用α1-AR的各个亚型抗体进行孵育，冲洗一抗后孵育荧光二抗，冲洗后经红外荧光成像，最后经Imagej软件分析各反应条带的光密度值（IOD）。各肌条中受体蛋白的相对含量为α1-AR各亚型与内参（GAPDH）的比值大小代表。 结果： 1RT-PCR结果显示，三种α1受体亚型的mRNA在各肌条均有表达：分别是α1A-AR, α1B-AR, α1D-AR。α1A-AR它在食管平滑肌、套索纤维、钩状纤维和胃底平滑肌表达水平分别为1.307±0.118；1.350±0.177；1.289±0.180；1.361±0.268； α1B-AR表达水平依次为1.222±0.151；1.279±0.237；1.309±0.263；1.352±0.267； α1D-AR表达水平依次为1.258±0.297；1.323±0.237；1.310±0.295；1.282±0.288；同一种肌条组织中，α1-AR的三种受体亚型的表达量无统计学差异(F=0.530, P=0.590)。同一种α1-AR受体亚型中，四种肌条间mRNA的表达无差异(F=0.037，P=0.910)。 2Western-blot结果显示，三种α1受体亚型均有其相应蛋白条带的表达：分别是α1A-AR, α1B-AR, α1D-AR。α1A-AR它在食管平滑肌、套索纤维、钩状纤维和胃底平滑肌表达水平分别为0.431±0.118；0.474±0.177；0.503±0.180；0.485±0.268； α1B-AR表达水平依次为0.335±0.151；0.458±0.237；0.459±0.263；0.465±0.267； α1D-AR表达水平依次为0.459±0.297；0.524±0.237；0.511±0.295；0.483±0.288；同一种肌条组织中，，α1-AR三种受体亚型的表达量无统计学差异(F=0.208, P=0.813)。同一种α1-AR受体亚型中，四种肌条间蛋白的表达无差异(F=0.188, P=0.905)。 结论：α1A-AR, α1B-AR, α1D-AR在人LES均表达。其表达水平在同种受体和同种肌条间无明显差异。可能在人LES的功能调节中发挥着作用。
[Abstract]:Objective: Primary esophageal motor dysfunction diseases such as achalasia, Nutcracker esophagus, hypertonic LES and so on are a kind of diseases that seriously affect people's quality of life. These diseases are often accompanied by some typical symptoms, such as dysphagia, etc. In recent years, with the improvement of people's living standards and esophageal manometry and 24-hour PH testing. However, the etiology and pathogenesis of these diseases are still unclear. Studies have shown that primary esophageal motor dysfunction is related to a variety of factors, including nerve, hormone regulation, psychosocial factors, inflammation, viral infection and so on. Autoimmune factors may be involved in the pathogenesis of primary esophageal motility disorders.
The human lower esophageal sphincter (LES) is located at the junction of the esophagus and stomach. Studies have shown that LES is composed of sling fibers (SF) and clasp fibres (CF). LES regulates the pressure of the lower esophagus with the help of the diaphragm foot under the regulation of various factors. Physiological significance. Valves play a key role in physiological anti-reflux. Disorders of their functions can lead to a variety of esophageal dyskinesia. The study of the regulatory mechanisms here is of great significance for gastroesophageal reflux diseases and esophageal dyskinesia.
Adrenergic alpha 1 receptor (alpha 1-AR) and its subtypes belong to G protein-coupled receptors, which are widely expressed in human and mammalian tissues. There has been no study on the expression, distribution and function of alpha 1-AR receptor and its subtypes in human LES.
In this study, Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of mRNA and protein receptors of alpha 1-AR and its receptor subtypes alpha 1A-AR, alpha 1B-AR and alpha 1D-AR in esophageal smooth muscle, Lasso fiber, hook fiber and gastric fundus smooth muscle. It provides a theoretical basis for the study of the function of alpha 1-AR receptor and the treatment of esophageal motility disorders.
Methods:Thirty patients with esophageal carcinoma underwent subtotal esophagectomy.Fresh specimens of esophagogastric junction were collected in the operating room.The specimens were carefully dissected from the ligature,uncinate,gastric fundus and esophageal circular muscle strips.Total RNA was retranscribed into cDNA and three primers of alpha-1-AR were used for RT-PCR. The relative content of mRNA in each muscle strip was represented by the ratio of each subtype of alpha-1-AR to internal reference (beta-actin). Total tissue protein was extracted, and the protein concentration was adjusted to the same concentration by BCA method. The subtypes of alpha-1-AR were separated by electrophoresis and then transferred to membranes. Imagej software was used to analyze the optical density (IOD) of each reaction band. The relative content of receptor protein in each muscle strip was represented by the ratio of each subtype of alpha 1-AR to internal reference (GAPDH).
Result:
The results of 1RT-PCR showed that three subtypes of alpha 1 receptor were expressed in all muscle strips: alpha 1A-AR, alpha 1B-AR and alpha 1D-AR.alpha 1A-AR. The expression levels of alpha 1 receptor in esophageal smooth muscle, Lasso fiber, hook fiber and gastric fundus smooth muscle were 1.307 [0.118], 1.350 [0.177], 1.289 [0.180], 1.361 [0.268], and 1.222 [0] respectively. There was no significant difference in the expression of three subtypes of alpha-1-AR receptor in the same muscle tissue (F = 0.530, P = 0.590). There was no difference (F=0.037, P=0.910).
Western-blot results showed that the three subtypes of alpha-1 receptor expressed corresponding protein bands: alpha 1A-AR, alpha 1B-AR, alpha 1D-AR.alpha 1A-AR in esophageal smooth muscle, Lasso fiber, uncinate fiber and gastric fundus smooth muscle, respectively, 0.431 [0.118], 0.474 [0.177], 0.503 [0.180], 0.485 [0.268], and the expression level of alpha 1B-AR was dependent on There was no significant difference in the expression of three subtypes of alpha-1-AR receptor in the same muscle strip (F = 0.208, P = 0.813). Among the four subtypes of alpha-1-AR receptor, there was no significant difference in the expression of three subtypes of alpha-1-AR receptor (F = 0.208, P = 0.813). There was no difference in protein expression (F=0.188, P=0.905).
CONCLUSION: Alpha 1A-AR, alpha 1B-AR and alpha 1D-AR are all expressed in human LES, and their expression levels are not significantly different between the same receptor and the same muscle strip.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R571
本文编号：2185954
[Abstract]:Objective: Primary esophageal motor dysfunction diseases such as achalasia, Nutcracker esophagus, hypertonic LES and so on are a kind of diseases that seriously affect people's quality of life. These diseases are often accompanied by some typical symptoms, such as dysphagia, etc. In recent years, with the improvement of people's living standards and esophageal manometry and 24-hour PH testing. However, the etiology and pathogenesis of these diseases are still unclear. Studies have shown that primary esophageal motor dysfunction is related to a variety of factors, including nerve, hormone regulation, psychosocial factors, inflammation, viral infection and so on. Autoimmune factors may be involved in the pathogenesis of primary esophageal motility disorders.
The human lower esophageal sphincter (LES) is located at the junction of the esophagus and stomach. Studies have shown that LES is composed of sling fibers (SF) and clasp fibres (CF). LES regulates the pressure of the lower esophagus with the help of the diaphragm foot under the regulation of various factors. Physiological significance. Valves play a key role in physiological anti-reflux. Disorders of their functions can lead to a variety of esophageal dyskinesia. The study of the regulatory mechanisms here is of great significance for gastroesophageal reflux diseases and esophageal dyskinesia.
Adrenergic alpha 1 receptor (alpha 1-AR) and its subtypes belong to G protein-coupled receptors, which are widely expressed in human and mammalian tissues. There has been no study on the expression, distribution and function of alpha 1-AR receptor and its subtypes in human LES.
In this study, Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of mRNA and protein receptors of alpha 1-AR and its receptor subtypes alpha 1A-AR, alpha 1B-AR and alpha 1D-AR in esophageal smooth muscle, Lasso fiber, hook fiber and gastric fundus smooth muscle. It provides a theoretical basis for the study of the function of alpha 1-AR receptor and the treatment of esophageal motility disorders.
Methods:Thirty patients with esophageal carcinoma underwent subtotal esophagectomy.Fresh specimens of esophagogastric junction were collected in the operating room.The specimens were carefully dissected from the ligature,uncinate,gastric fundus and esophageal circular muscle strips.Total RNA was retranscribed into cDNA and three primers of alpha-1-AR were used for RT-PCR. The relative content of mRNA in each muscle strip was represented by the ratio of each subtype of alpha-1-AR to internal reference (beta-actin). Total tissue protein was extracted, and the protein concentration was adjusted to the same concentration by BCA method. The subtypes of alpha-1-AR were separated by electrophoresis and then transferred to membranes. Imagej software was used to analyze the optical density (IOD) of each reaction band. The relative content of receptor protein in each muscle strip was represented by the ratio of each subtype of alpha 1-AR to internal reference (GAPDH).
Result:
The results of 1RT-PCR showed that three subtypes of alpha 1 receptor were expressed in all muscle strips: alpha 1A-AR, alpha 1B-AR and alpha 1D-AR.alpha 1A-AR. The expression levels of alpha 1 receptor in esophageal smooth muscle, Lasso fiber, hook fiber and gastric fundus smooth muscle were 1.307 [0.118], 1.350 [0.177], 1.289 [0.180], 1.361 [0.268], and 1.222 [0] respectively. There was no significant difference in the expression of three subtypes of alpha-1-AR receptor in the same muscle tissue (F = 0.530, P = 0.590). There was no difference (F=0.037, P=0.910).
Western-blot results showed that the three subtypes of alpha-1 receptor expressed corresponding protein bands: alpha 1A-AR, alpha 1B-AR, alpha 1D-AR.alpha 1A-AR in esophageal smooth muscle, Lasso fiber, uncinate fiber and gastric fundus smooth muscle, respectively, 0.431 [0.118], 0.474 [0.177], 0.503 [0.180], 0.485 [0.268], and the expression level of alpha 1B-AR was dependent on There was no significant difference in the expression of three subtypes of alpha-1-AR receptor in the same muscle strip (F = 0.208, P = 0.813). Among the four subtypes of alpha-1-AR receptor, there was no significant difference in the expression of three subtypes of alpha-1-AR receptor (F = 0.208, P = 0.813). There was no difference in protein expression (F=0.188, P=0.905).
CONCLUSION: Alpha 1A-AR, alpha 1B-AR and alpha 1D-AR are all expressed in human LES, and their expression levels are not significantly different between the same receptor and the same muscle strip.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R571
【参考文献】
相关期刊论文 前4条
1 高立平;刘俊峰;温士旺;田子强;季保庆;王其彰;;胆囊收缩素受体对人食管下括约肌套索纤维和钩状纤维的调节机制[J];中华实验外科杂志;2006年04期
2 温士旺;刘俊峰;高立平;田子强;王其彰;李保庆;;一氧化氮介导人食管下括约肌舒张的作用机制[J];中华医学杂志;2006年01期
3 高立平;刘俊峰;温士旺;;胃泌素和胃动素对人离体食管下括约肌张力的调节机制[J];基础医学与临床;2006年03期
4 温士旺;刘俊峰;高立平;李保庆;王福顺;王其彰;田子强;;毒蕈碱受体亚型对人食管下括约肌的调节作用[J];中国药理学通报;2008年07期
本文编号：2185954
本文链接：https://www.wllwen.com/yixuelunwen/xiaohjib/2185954.html
最近更新
教材专著
