NMDA谷氨酸受体NR1亚基在大鼠前庭终器的表达

目的研究NMDA谷氨酸受体NR1亚基在大鼠前庭终器的表达。方法运用RT-PCR技术检测大鼠前庭终器NR1亚基的基因表达。以大鼠脑组织RNA为阳性对照。结果在前庭终器中扩增出标志NMDA谷氨酸受体NR1亚基基因表达的PCR产物,并经测序证实与预计扩增的cDNA片断序列一致。结论NMDA谷氨酸受体NR1亚基在大鼠前庭终器有表达,为谷氨酸作为前庭重要的神经递质之一提供了证据。NMDA谷氨酸受体可能作为自身受体在前庭系统神经信息传递过程中起到正反馈调节作用。     

γ-氨基丁酸受体γ1亚基在大鼠前庭终器的表达

目的：研究γ-氨基丁酸受体γ1亚基在大鼠前庭终器的表达。方法；运用RT-PCR技术检测大鼠前庭终器编码GABAA受体亚基因表达。以大鼠脑组织RNA为阳性对照。结果：扩增出标志GABAA受体基因表达的PCR产物。结论:GABA受体γ1亚基在大鼠前庭终器有表达，为证明GABA是前庭系统重要神经递质之一提供了支持。     

N型乙酰胆碱受体α10亚基在大鼠内耳耳蜗及前庭中的表达

目的研究N型乙酰胆碱受体α10亚基在大鼠前庭终器、Scarpa’s神经节、基底膜、螺旋神经节中的表达。方法运用RT-PCR技术分别检测大鼠前庭终器、Scarpa’s神经节、基底膜、螺旋神经节编码N型乙酰胆碱受体α10亚基的基因表达。以大鼠脑组织RNA为阳性对照。结果N型乙酰胆碱受体α10亚基在大鼠前庭终器、基底膜上有表达,在Scarpa’s神经节、螺旋神经节中无表达。结论在前庭终器和基底膜上扩增出标志N型乙酰胆碱受体α10亚基基因表达的PCR产物,为乙酰胆碱是内耳重要的神经递质之一提供了支持,并为N型乙酰胆碱受体在内耳中的传入-传出调节中的作用提供了依据。     

CABA_A受体亚单位在前庭系统的表达

-氨基丁酸(GABA)是哺乳类动物中枢神经系统最丰富和功能最重要的抑制性神经递质。GABA受体有两种不同类型—GABAA受体和GABAB受体。近来的研究发现哺乳类动物壶腹嵴、球囊和前庭传入神经胞体有GABAA受体亚单位的表达。为确定GABAA受体亚单位的mRNA,该作者使用基因特异性引物反转录PCR技术分析研究大鼠前庭终器GABAA的13种受体亚单位mRNA的表达。结果显示:PCR扩增产物代表GABAA受体亚单位基因α1～6、β1～3、1～3得到表达,其中α5、α6、β3扩增后仅产生微弱表达,经过再扩增后得到显示,而亚单位δ未得到表达。对照试验…     

癌-睾丸抗原SCP-1 mRNA在人鼻咽癌中的表达

目的：从基因水平了解癌-睾丸抗原SCP-1在人鼻咽癌（NPC）的表达情况。方法：用逆转录-聚合酶链反应（RT—PCR）技术，检测28例NPC组织和40例非癌鼻咽部组织SCP-1 mRNA的表达。随机抽取SCP-1表达阳性的PCR产物进行DNA测序并结合临床指标进行统计学处理。结果：SCP-1 mRNA在NPC的表达率为14．3％（4／28），在非癌鼻咽部组织无表达；测序结果表明PCR扩增产物序列与目的基因序列相符；SCP-1 mRNA的表达率与NPC患者的年龄、性别和血清抗EB病毒水平无相关性（P〉0．05）。结论：SCP-1 mRNA在NPC组织中有表达，提示SCP-1可能是一种新的NPC肿瘤靶抗原。     

大前庭水管综合征PDS基因突变检测

目的 检测大前庭水管综合征患者PDS基因突变位点,从遗传分子水平探讨该病的发病机制.方法收集18例散发大前庭水管综合征患者和12例听力正常健康对照个体外周血DNA样本共30份;PCR扩增研究对象PDS基因第6、9外显子,对PCR扩增产物直接测序分析.结果全部受检对象PDS基因第6、9外显子PCR扩增均获成功,测序分析发现1 8例大前庭水管综合征患者中1例PDS基因编码区611G→C和612T→G颠换,导致204密码子错义突变;12例听力正常者未发现任何突变位点.结论大前庭水管综合征患者存在PDS基因突变,对大前庭水管综合征PDS基因其它部位序列的研究,有助进一步测序分析揭示该病的实质.     

浆膜蛋白RTN1和RTN4基因在小鼠内耳的表达

目的 探讨浆膜蛋白RTN1和RTN4基因在小鼠内耳的表达。方法 采用5只成年小鼠内耳组织提取总RNA，逆转录后获得小鼠内耳细胞cDNA，根据RTN1和RTN4基因编码区序列设计的引物进行PCR扩增，通过PCR产物分析和DNA测序确定RTN1和RTN4是否在小鼠内耳细胞表达。结果 采用小鼠内耳组织总RNA，RT—PCR扩增出RTN1和RTN4基因部分编码区，扩增产物测序证实小鼠内耳中有RTN1和RTN4基因的表达。结论 RTN1和RTN4基因在内耳有表达，为RTN1和RTN4与连接蛋白26（connexin26）蛋白的互作关系提供了进一步的证据。浆膜蛋白RTN1和RTN4可能与连接蛋白26在听觉生理中起作用。     

单侧迷路切除后γ-氨基丁酸A受体α1亚单位mRNA在大鼠小脑绒球中的表达

目的：阐明大鼠小脑绒球中γ-氨基丁酸A（GABAA）受体α1亚单位在前庭代偿过程中的作用。方法：以实时定量PCR技术对迷路切除后GABAA受体α1亚单位mRNA在前庭代偿早期大鼠小脑绒球中的表达进行研究。结果：GABAA受体α1亚单位在小脑绒球中有表达，但迷路切除后在损伤同侧和对侧绒球中其表达无统计学意义。结论：大鼠小脑绒球中存在GABAA受体α1亚单位，在前庭代偿的早期，GABAA受体α1亚单位mRNA表达的变化极可能没有涉及前庭代偿。     

鼻息肉组织中白细胞介素5mRNA表达状况的半定量分析

目的 观察鼻息肉组织中白细胞介素5(Interleukin-5,IL-5)mRNA的表达水平。方法 采用逆转录PCR(RT-PCR)法,半定量检测鼻息肉(11例)和正常中鼻甲(8例)组织中IL-5 mRNA表达水平。结果 鼻息肉中IL-5 mRNA表达状况的半定量分析显示,11例鼻息肉标本RT-PCR产物中均可见IL-5和β-actin的扩增条带,IL-5/β-actin光密度比值0.711±0.112,IL-5 mRNA阳性表达率为100％。而8例正常中鼻甲组织的扩增产物中只有1例显示两条条带,余者均只有β-actin条带,IL-5 mRNA阳性表达率为12.5％。结论 鼻息肉中IL-5 mRNA阳性表达水平高于正常鼻粘膜组织,提示鼻息肉组织中IL-5mRNA的表达水平在鼻息肉的发病机制中发挥作用。     

原位杂交定位检测大鼠前庭末梢γ—氨基丁酸A受体亚单位α2mRNA

目的 了解γ－氨基丁酸（γ－aminobutynric acid,GABA)在前庭末梢神经传递中的作用。方法 采用大鼠内耳石蜡切片，地高辛标记的GABAAα2cDNA探针（549个碱基），抗地高碱性磷酸酶检测系统，BM Purple AP Substrate显色系统，原位杂交（in-situ hybridization)检测大鼠前庭末梢GABAα2mRNA受体的表达。结果 所有的大鼠前庭神经节细胞体及壶腹嵴上围绕I型毛细胞的神经杯状物表达GABAA受体α2亚单位mRNA。毛细胞和支持细胞不表达。作阳性组织对照的大鼠小脑皮质蒲肯野氏细胞和颗粒细胞的胞体和触突表达GABAAα2。OMP探针阴性对照，无探针的空白对照及无地高辛抗体空白对照的内耳及小脑切片均不表达GABAAα2。结论 GABAAα2受体位于前庭的传入神经末端和其胞体，强烈支持GABA作为前庭末梢的传入神经递质的结论。并提示其在前庭传入神经传递中具有重要作用。     

Expression of G-protein Alpha Subunit Genes in the Vestibular Periphery of Rattus norvegieus and their Chromosomal Mapping

Objective—Heterotrimeric G-proteins play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. The precise role of G-proteins and their coupled receptors in the physiology of the vestibular neuroepithelium is not well understood. The purpose of this study was to better define the role of these proteins by examining their expression in the rat vestibular periphery and characterizing their chromosomal location.

Material and Methods—To characterize G-protein α subunit gene expression in the target tissue of interest, we performed polymerase chain reaction (PCR) using degenerate G-protein primers corresponding to conserved regions in the G-protein α subunit coding sequence on a normalized rat vestibular cDNA library. PCR amplicons were cloned and 50 clones were randomly selected and sequenced. Radiation hybrid (RH) mapping was used to determine the chromosomal location of Gα_olf and two previously identified G-protein α subunits—Gα_i2 and Gα_i2(vest)—in the rat genome.

Results—The following G-protein α subunits were identified in the normalized cDNA library: Gα_olf, Gα_s, Gα_o and Gα_s2. Gα_olf maps to chromosome 18 between markers D18Mit17b and D18Mgh2. Gα_i2 maps to chromosome 8 between markers D8Rat65 and D8Mgh2. Gα_i2(vest) maps to chromosome 1 between markers D1Rat132 and D1Rat202. These chromosomal locations in the rat genome are syntenic to chromosomal regions in which the homologous G-protein α subunit genes have been localized in the human and mouse genomes, further validating RH mapping as an effective and accurate tool. We were unable to RH map the location of Gα_o due to its extensive homology with the hamster gene.

Conclusion—The characterization of G-protein alpha subunit gene expression in the vestibular periphery and the chromosomal localization of these genes in the rat revealed that a diverse group of these second messengers are expressed.     

Radiation hybrid mapping of five muscarinic acetylcholine receptor subtype genes in Rattus norvegicus

Acetylcholine is the main neurotransmitter of the vestibular efferent system and a wide variety of muscarinic and nicotinic acetylcholine receptors are expressed in the vestibular periphery. The role of these receptors and in particular the role of muscarinic acetylcholine receptors in the physiology of the vestibular neuroepithelium is not understood. Congenic and consomic rats are a convenient way to investigate the involvement of candidate genes in the manifestation of defined traits. To use congenic or consomic rats to elucidate the roles of these receptors in vestibular physiology or pathology the chromosomal location of the genes encoding these receptors has to be determined. Using radiation hybrid (RH) mapping and a rat RH map server (www.rgd.mcw.edu/RHMAP SERVER/), we determined the chromosomal locations of the muscarinic acetylcholine receptor genes in the rat (Rattus norvegicus). The m1-m5 muscarinic subtypes mapped to the following chromosomes: Chrm1, chromosome 1; Chrm2, chromosome 4; Chrm3, chromosome 17; Chrm4, chromosome 3; and Chrm5, chromosome 3. With the chromosomal location for each of these muscarinic subtypes known, it is now possible to develop congenic and consomic strains of rats that can be used to study the functions of each of these subtypes.     

Type 1 vanilloid receptor expression by mammalian inner ear ganglion cells

The type 1 vanilloid receptor (VR1) is a non-specific cation channel activated by capsaicin, lipoxygenase (LOX) products, heat and acid. This study demonstrates VR1 and 5-LOX expression by inner ear ganglion cells. A PCR product (210 bp) was amplified from both oligo(dT)- and random primer-generated cDNAs of rat spiral ganglion cells using VR1 gene-specific primers constructed from the 3' non-homologous region. This PCR product shared 100% sequence homology to a rat VR1 cDNA (GenBank accession no. AF029310) and a rat vanilloid receptor splice variant mRNA (GenBank accession no. AF158248). Frozen sections of PLP-fixed, decalcified Long-Evans rat temporal bones were stained immunohistochemically for VR1. Neurons and satellite cells in both the vestibular and spiral ganglia were VR1-immunopositive. Neurons and supporting cells in adjacent sections of these ganglia were immunopositive for 5-LOX. These findings raise the hypothesis that activation of VR1 by endogenous ligands may contribute to hypersensitivity of the eighth nerve to hair cell inputs in a variety of pathologic conditions, such as tinnitus, Meniere's disease and migraine. In particular, these data suggest that LOX activation during inflammatory processes or during cyclo-oxygenase inhibition (e.g. by aspirin) is a potential intrinsic source of VR1 activation in inner ear ganglia.     

Comparison of the vascular innervation of the rat cochlea and vestibular system

In order to gain a better understanding of the neuronal and local control of inner ear blood flow, the vascular innervation to the rat cochlea and vestibular system was examined. Specimens were removed in toto beginning at the basilar artery extending to the anterior inferior cerebellar artery, labyrinthine artery, common cochlear artery, modiolar artery and anterior vestibular artery. When possible the vessels were dissected in continuity through the cribrose area. The vestibular endorgans were also removed. Specimens were examined using immunohistochemical techniques for the presence of vasoactive intestinal peptide, neuronal nitric oxide synthase, neuropeptide-Y, substance P and calcitonin gene related peptide. Results show that the vasculature to the cochlea and vestibular portion of the inner ear receive similar types of nonadrenergic innervation, that within the vestibular endorgans, only CGRP and SP were found in the neuroepithelium or in association with vessels, and that within the vestibular system, the majority of the vascular innervation appears to stop at or near the cribrose area. In the cochlea however, it extends to include the radiating arterioles. These findings suggest that cochlear blood flow is under finer control and that neuronally induced changes in blood flow may have a more global effect in the vestibular periphery.