M型乙酰胆碱受体在豚鼠前庭中的表达的研究

发布时间：2018-05-20 10:23

本文选题：M型乙酰胆碱受体 + 前庭终器　；参考：《华中科技大学》2011年博士论文

【摘要】：第一部分M型乙酰胆碱受体蛋白在豚鼠前庭中的表达的实验研究 [目的]mAChRs在中枢和外周神经系统中广泛表达,并在调节细胞活动和功能中起着重要的作用。乙酰胆碱(acetylcholine, ACh)是哺乳动物主要的内耳传出神经递质,在外周前庭系统中存在各种毒菌碱型乙酰胆碱受体(muscarinic acetylcholine receptor, mAChR)和烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR)。目前的研究表明M1-M5型乙酰胆碱受体在哺乳动物前庭终器中均有表达,但这些受体蛋白在豚鼠前庭中的表达尚无报道,本实验研究编码M型乙酰胆碱受体(muscarinic acetylcholine receptor, mAChR)的蛋白在豚鼠前庭终器中的表达。 [方法]从豚鼠内耳中分离出前庭组织分别提取半规管(水平半规管,垂直半规管,后半规管),球囊,椭圆囊蛋白,应用western blot法检测M1-M5 mAChR蛋白在以上前庭终器中的表达。 [结果]在豚鼠前庭终器(半规管,椭圆囊,球囊)中均可检测到M1-M5 mAChR蛋白的表达。 [结论]本研究运用western blot的方法成功的检测出M1-M5 mAChR蛋白在豚鼠前庭终器(半规管,椭圆囊,球囊)中的表达,由此为M1-M5 mAChR在豚鼠前庭中的存在提供证据,并证实M1-M5 mAChR也可能在介导前庭毛细胞胆碱能传出过程中起到重要的作用,进而为前庭疾病机制阐明及前庭疾病的治疗提供理论基础。第二部分M型乙酰胆碱受体在豚鼠前庭终器中的表达的实验研究 [目的]目前研究证实乙酰胆碱(acetylcholine, ACh)在外周前庭系统中广泛表达,在哺乳动物内耳中存在各种毒菌碱型乙酰胆碱受体(muscarinic acetylcholine receptor, mAChR)和烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR),是哺乳动物内耳的主要传出神经递质。目前的研究表明M1-M5型乙酰胆碱受体在哺乳动物前庭终器中均有表达,但这些受体在豚鼠前庭组织中的表达尚无报道,本实验研究编码M1-M5型乙酰胆碱受体(muscarinic acetylcholine receptor, mAChR)的mRNA在豚鼠前庭终器(半规管,椭圆囊,球囊)中的表达。 [方法]从豚鼠内耳中分离出前庭组织并提取RNA,采用逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)法检测编码M1-M5mAChR的mRNA是否在豚鼠前庭组织中有表达。 [结果]在豚鼠前庭终器中可检测到编码M1-M5 mAChR基因的mRNA的表达,经测序证实其与预计扩增的cDNA序列一致。 [结论]本研究运用RT-PCR的方法成功的检测出编码M1-M5 mAChR基因的mRNA在豚鼠前庭终器中的表达,由此为M1-M5 mAChR在豚鼠VHCsⅡ中的存在提供证据,并证实M1-M5 mAChR也可能在介导前庭毛细胞胆碱能传出过程中起到重要的作用,进而为前庭疾病机制阐明及前庭疾病的治疗提供理论基础。第三部分m型乙酰胆碱受体在豚鼠前庭毛细胞中的表达的实验研究 [目的]目前研究证实乙酰胆碱(acetylcholine, ACh)在外周前庭系统中广泛表达,在哺乳动物内耳中存在各种毒菌碱型乙酰胆碱受体(muscarinic acetylcholine receptor, mAChR)和烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR),是哺乳动物内耳的主要传出神经递质。目前的研究表明M1-M5型乙酰胆碱受体在哺乳动物前庭终器中均有表达,但这些受体在豚鼠前庭毛细胞中的表达国内外尚无报道,本实验研究编码M1-M5型乙酰胆碱受体(M1-M5 muscarinic acetylcholine receptor, M1-M5 mAChR)的mRNA在豚鼠前庭终器及Ⅱ型前庭毛细胞(vestibular hair cell, VHC)中的表达。 [方法]从豚鼠前庭组织中分离出Ⅱ型前庭毛细胞,在显微操作仪器下应用玻璃电极捕获单个Ⅱ型前庭毛细胞,应用单细胞RT-PCR(single cell reverse transcription polymerase chain reaction, SC-RT-PCR)法检测编码M1-M5 mAChR的mRNA是否在豚鼠Ⅱ型前庭毛细胞中有表达,从而明确M1-M5 mAChR在前庭组织中的细胞定位。 [结果]编码M1-M5 mAChR基因的mRNA在豚鼠Ⅱ型前庭毛细胞中均可被检测到,经测序证实其与预计扩增的cDNA序列一致。 [结论]本研究运用单细胞RT-PCR的方法检测编码M1-M5 mAChR的mRNA在豚鼠VHCsⅡ中的表达,由此为M1-M5 mAChR在豚鼠VHCsⅡ中的存在提供证据,并证实M1-M5mAChR也可能在介导前庭毛细胞胆碱能传出过程中起到重要的作用,进而为前庭疾病机制阐明及前庭疾病的治疗提供理论基础。
[Abstract]:Part one: Experimental Study on the expression of M acetylcholine receptor protein in the vestibule of guinea pigs
[Objective]mAChRs is widely expressed in the central and peripheral nervous systems and plays an important role in regulating cell activity and function. Acetylcholine (ACh) is the main mammalian inner ear efferent neurotransmitter, and there are various virulent alkali acetylcholine receptors (muscarinic acetylcholine receptor) in the peripheral vestibule system. MAChR) and nicotinic acetylcholine receptor (nicotinic acetylcholine receptor, nAChR). Current studies have shown that M1-M5 type acetylcholine receptors are expressed in mammalian vestibule terminals, but the expression of these receptor proteins in the vestibule of guinea pigs is not reported. This experiment studies the encoding of M acetylcholine receptor (muscarinic acetylcholine). The expression of receptor, mAChR protein in the vestibular end organ of guinea pigs.
[Methods] the semicircular canals (horizontal semicircular canals, vertical semicircular canals, posterior semicircular canals), balloon and oval cysts were isolated from the inner ear of guinea pigs and detected the expression of M1-M5 mAChR protein in the above vestibular end organs by Western blot method.
[results] the expression of M1-M5 mAChR protein could be detected in the vestibular terminal organs (semicircular canal, utricle and balloon) of guinea pigs.
[Conclusion] this study successfully detected the expression of M1-M5 mAChR protein in the vestibular terminal (semicircular canals, oval sac and balloon) of guinea pigs by Western blot, which provided evidence for the existence of M1-M5 mAChR in the vestibule of guinea pigs, and confirmed that M1-M5 mAChR may also play an important role in mediating the cholinergic efferent of the vestibule hair cells. This will provide a theoretical basis for elucidating the mechanism of vestibular diseases and the treatment of vestibular diseases.
The second part is the experimental study of the expression of M acetylcholine receptor in the vestibular end organ of guinea pigs.
[Objective] the present study confirms that acetylcholine (ACh) is widely expressed in the peripheral vestibule system. There are various kinds of muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nicotinic acetylcholine receptor, nAChR) in mammalian inner ear, which are the inner ear of mammalian. The present study showed that the M1-M5 acetylcholine receptor was expressed in the mammalian vestibular end organ, but the expression of these receptors in the vestibule tissues of the guinea pig was not reported. This experiment studied the mRNA of the M1-M5 type acetylcholine receptor (muscarinic acetylcholine receptor, mAChR) in the vestibule end organ of the guinea pig. The expression in the tube, the oval sac, the balloon.
[Methods] vestibule tissue was isolated from the inner ear of guinea pig and RNA was extracted, and the expression of mRNA in the vestibule tissues of guinea pig was detected by reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR).
[results] the expression of mRNA encoding M1-M5 mAChR gene could be detected in the vestibular end organ of guinea pigs, and it was confirmed by sequencing that it was consistent with the predicted cDNA sequence.
[Conclusion] this study successfully detected the expression of mRNA encoding M1-M5 mAChR gene in the vestibular end organ of guinea pigs by using RT-PCR method, which provided evidence for the existence of M1-M5 mAChR in the guinea pig VHCs II, and confirmed that M1-M5 mAChR may also play an important role in mediating the cholinergic efferent of vestibular hair cells, and then the vestibular disease is a vestibular disease. Elucidating the pathogenesis and providing a theoretical basis for the treatment of vestibular diseases.
The third part is the experimental study of the expression of M acetylcholine receptor in guinea pig vestibular hair cells.
[Objective] the present study confirms that acetylcholine (ACh) is widely expressed in the peripheral vestibule system. There are various kinds of muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nicotinic acetylcholine receptor, nAChR) in mammalian inner ear, which are the inner ear of mammalian. The present study shows that the M1-M5 acetylcholine receptor is expressed in the mammalian vestibular end organ, but the expression of these receptors in the vestibular hair cells of the guinea pig is not yet reported. This experiment studies the mRNA of the M1-M5 muscarinic acetylcholine receptor (M1-M5 mAChR) encoding the M1-M5 type acetylcholine receptor in this experiment. Expression of vestibular and hair cell (VHC) in the vestibular end organ of guinea pigs.
[Methods] the type II vestibular hair cells were isolated from the vestibular tissue of guinea pigs, and the single type II vestibular hair cells were captured with glass electrodes under the micromanipulation instrument. The single cell RT-PCR (single cell reverse transcription polymerase chain reaction, SC-RT-PCR) method was used to detect whether the mRNA in the vestibule of M1-M5 mAChR was in the vestibular capillaries of the guinea pig type II. The expression of M1-M5 mAChR in the vestibular tissue is clear.
[results] mRNA encoding M1-M5 mAChR gene could be detected in guinea pig type II vestibular hair cells, and it was confirmed by sequencing that it was consistent with the predicted cDNA sequence.
[Conclusion] the present study used the single cell RT-PCR method to detect the expression of the mRNA encoding M1-M5 mAChR in the guinea pig VHCs II, thus providing evidence for the existence of M1-M5 mAChR in the guinea pig VHCs II, and confirmed that M1-M5mAChR may also play an important role in mediating the cholinergic efferent of vestibular hair cells, and further elucidate the mechanism of vestibular disease. It provides a theoretical basis for the treatment of vestibule disease.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764

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