腎上腺素α1受體在人食管下括約肌的表達研究
發(fā)布時間：2018-08-16 12:15
【摘要】：目的：原發(fā)性食管運動功能障礙性疾病如賁門失弛緩癥、胡桃夾食管、高張性LES等是一類嚴重影響人們生活質(zhì)量的疾病。這類疾病往往伴有一些典型癥狀，如下咽困難等。近些年來，隨著人群生活水平提高和食管測壓和24小時PH檢測等檢查儀器的應(yīng)用，原發(fā)性食管運動功能障礙性疾病的發(fā)現(xiàn)病例有所升高。但有關(guān)此類疾病的病因和發(fā)病機制尚不完全明確。研究顯示，原發(fā)性食管運動功能障礙性疾病和多種因素有關(guān)。神經(jīng)、激素調(diào)節(jié)、精神心理因素、炎癥反應(yīng)、病毒感染及自身免疫因素等均可能參與了原發(fā)性食管運動功能障礙性疾病的發(fā)病。 人食管下括約肌(Lower esophageal sphincter, LES)位于食管胃連接部。研究顯示，LES由套索纖維（sling fibers，SF）和鉤狀纖維(clasp fibres，CF)組成。其在機體多種因素的調(diào)節(jié)下協(xié)同膈腳調(diào)節(jié)食管下段腔內(nèi)壓。這個位于食管胃連接部的高壓帶具有重要的生理意義。如同閥門樣的作用使其在生理抗反流中發(fā)揮關(guān)鍵作用。而其功能的紊亂可導(dǎo)致多種食管運動功能障礙性疾病。研究此處的調(diào)節(jié)機制對胃食管返流類疾病和食管運動功能障礙性疾病具有重要意義。 腎上腺素α1受體（α1-AR）及其亞型屬于G蛋白偶聯(lián)受體，在人和哺乳動物體內(nèi)廣泛表達。現(xiàn)有研究提示，α1-AR對于平滑肌收縮的調(diào)節(jié)較為重要，但既往關(guān)于α1-AR表達的研究主要集中于血管、生殖泌尿、肝臟和腎臟等器官。對于胃腸道研究較少。尚未有對人LES的α1-AR受體及其亞型表達分布和功能的研究。 本實驗通過蛋白印跡法（Western-blot）和逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)來研究α1-AR及其受體亞型α1A-AR, α1B-AR, α1D-AR的mRNA和蛋白受體在食管平滑肌、套索纖維、鉤狀纖維和胃底平滑肌的表達水平。通過本項研究完善LES受體的表達規(guī)律，為此后α1-AR受體功能的研究和治療食管運動功能障礙性疾病提供理論基礎(chǔ)。 方法：選取因高位食管癌行食管大部切除術(shù)的患者30例。在手術(shù)室收集新鮮食管胃結(jié)合部標本，仔細游離套索纖維肌條、鉤狀纖維肌條、胃底和食管環(huán)形肌肌條。提取組織總RNA后反轉(zhuǎn)錄為cDNA，應(yīng)用3種α1-AR的引物行RT-PCR。分析擴增產(chǎn)物中目的條帶的光密度值(IOD)。各肌條中mRNA的相對含量為α1-AR各亞型與內(nèi)參（β-actin）的比值大小代表。提取組織總蛋白，BCA法測量蛋白濃度后將蛋白調(diào)整至相同濃度，電泳分離出α1-AR個亞型后轉(zhuǎn)膜，再分別應(yīng)用α1-AR的各個亞型抗體進行孵育，沖洗一抗后孵育熒光二抗，沖洗后經(jīng)紅外熒光成像，最后經(jīng)Imagej軟件分析各反應(yīng)條帶的光密度值（IOD）。各肌條中受體蛋白的相對含量為α1-AR各亞型與內(nèi)參（GAPDH）的比值大小代表。 結(jié)果： 1RT-PCR結(jié)果顯示，三種α1受體亞型的mRNA在各肌條均有表達：分別是α1A-AR, α1B-AR, α1D-AR。α1A-AR它在食管平滑肌、套索纖維、鉤狀纖維和胃底平滑肌表達水平分別為1.307±0.118；1.350±0.177；1.289±0.180；1.361±0.268； α1B-AR表達水平依次為1.222±0.151；1.279±0.237；1.309±0.263；1.352±0.267； α1D-AR表達水平依次為1.258±0.297；1.323±0.237；1.310±0.295；1.282±0.288；同一種肌條組織中，α1-AR的三種受體亞型的表達量無統(tǒng)計學(xué)差異(F=0.530, P=0.590)。同一種α1-AR受體亞型中，四種肌條間mRNA的表達無差異(F=0.037，P=0.910)。 2Western-blot結(jié)果顯示，三種α1受體亞型均有其相應(yīng)蛋白條帶的表達：分別是α1A-AR, α1B-AR, α1D-AR。α1A-AR它在食管平滑肌、套索纖維、鉤狀纖維和胃底平滑肌表達水平分別為0.431±0.118；0.474±0.177；0.503±0.180；0.485±0.268； α1B-AR表達水平依次為0.335±0.151；0.458±0.237；0.459±0.263；0.465±0.267； α1D-AR表達水平依次為0.459±0.297；0.524±0.237；0.511±0.295；0.483±0.288；同一種肌條組織中，，α1-AR三種受體亞型的表達量無統(tǒng)計學(xué)差異(F=0.208, P=0.813)。同一種α1-AR受體亞型中，四種肌條間蛋白的表達無差異(F=0.188, P=0.905)。 結(jié)論：α1A-AR, α1B-AR, α1D-AR在人LES均表達。其表達水平在同種受體和同種肌條間無明顯差異�？赡茉谌薒ES的功能調(diào)節(jié)中發(fā)揮著作用。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R571
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R571
【參考文獻】
相關(guān)期刊論文 前4條
1 高立平;劉俊峰;溫士旺;田子強;季保慶;王其彰;;膽囊收縮素受體對人食管下括約肌套索纖維和鉤狀纖維的調(diào)節(jié)機制[J];中華實驗外科雜志;2006年04期
2 溫士旺;劉俊峰;高立平;田子強;王其彰;李保慶;;一氧化氮介導(dǎo)人食管下括約肌舒張的作用機制[J];中華醫(yī)學(xué)雜志;2006年01期
3 高立平;劉俊峰;溫士旺;;胃泌素和胃動素對人離體食管下括約肌張力的調(diào)節(jié)機制[J];基礎(chǔ)醫(yī)學(xué)與臨床;2006年03期
4 溫士旺;劉俊峰;高立平;李保慶;王福順;王其彰;田子強;;毒蕈堿受體亞型對人食管下括約肌的調(diào)節(jié)作用[J];中國藥理學(xué)通報;2008年07期
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