神经降压素受体1在先天性巨结肠症肠壁肌间神经丛的表达及意义

发布时间：2017-12-31 06:38

  本文关键词：神经降压素受体1在先天性巨结肠症肠壁肌间神经丛的表达及意义　出处：《广州医科大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 先天性巨结肠 NTSR1 免疫组化 肌间神经丛 肠神经系统

【摘要】：目的先天性巨结肠症(Hirschsprung’s disease,HSCR)又称“肠壁无神经节细胞症”,是由遗传、环境等多种因素共同作用导致肠神经系统(Enteric nervous system,ENS)早期发生多基因突变或表达异常的神经嵴细胞源性的一种先天性肠道疾病。其基本病理生理改变为远端病变结肠黏膜下神经丛(Submucosal plexuses)和肌间神经丛(Myenteric plexuses)内完全或部分缺乏肠神经节细胞(Intestinal ganglion cells,IGCS),致使神经纤维排列紊乱,呈波浪状,造成结肠持续痉挛,失去正常的推进蠕动功能。由于粪便通过困难,长期淤积于痉挛肠段,近端结肠代偿性扩张、增厚,形成巨结肠。ENS是一个复杂而积极的胃肠道动力和感觉神经系统,由肠道肌间和黏膜下神经丛共同构成,两者之间紧密联系,对胃肠道的运动、分泌功能具有独立的调节作用。HSCR是ENS早期发生基因突变或功能丧失的结果。神经降压素(neurotensin,NT)是由肠道具有特殊内分泌功能的开放型的N细胞分泌的一种神经递质或调质,属于配体依赖性的转录因子,在中枢神经系统(Central nervous system,CNS)以及心血管、呼吸、消化、内分泌、免疫等多种外周系统中存在并发挥重要的功能调节作用。神经降压素受体1(Neurotensin receptor 1,NTSR1)是一种具有7个跨膜结构域的G蛋白偶联受体,在人体结肠中与NT配基亲和力最高。NT通过介导G蛋白偶联受体NTSR1激活相应的信号通路,产生向下的信号传递,到达胃肠道平滑肌等效应部位,从而增强结肠的推进运动,使粪便排空。NTSR1在ENS中对肠道动力具有十分重要的作用。在我们的前期研究中,利用基因芯片技术检测出大量的HSCR差异性表达基因,其中ntsr1基因表达明显下调,并经rt-pcr验证。查阅相关文献,目前国内外在nt-ntsr1的相互作用与hscr发病的相关关系方面研究较少。有研究已发现,ntsr1在人体正常结肠组织黏膜下和肌间神经丛存在不同程度的阳性表达,而ntsr1在hscr各肠段是否表达并存在表达的异常尚不清楚。因此,本研究从前期研究结果中选取ntsr1基因,采用免疫组织化学染色法观察ntsr1在hscr各段肠壁肌间神经丛的表达情况,从而初步探讨ntsr1与hscr发病的可能关系。方法选取2010年1月至2014年9月在广东省妇幼保健院小儿外科经肛门巨结肠根治术治疗的hscr患儿手术标本42例作为实验组,实验组分为狭窄段、移行段、扩张段、正常段四个组别。所有患儿均为首次接受手术治疗,均为散发病例,无合并其他主要脏器的先天性畸形或综合症,近系亲属均无hscr病例。所有患儿均根据术前临床表现、钡剂灌肠造影等检查,以及术中快速冰冻病理及术后病理科医师二次he染色诊断结果综合确诊。所选取病例符合正态分布的原则。选取与实验组年龄相近的如先天性无肛门、结肠穿孔、乙状结肠冗长症等非hscr患儿手术切除的正常结肠标本20例作为对照组,对照组家族中无hscr病例及其它肠神经发育异常性疾病。在光学显微镜下观察实验组与对照组病例he染色的结果并统计。应用免疫组织化学染色法检测42例实验组标本狭窄段、移行段、扩张段、正常段及20例正常对照组标本肠壁肌间神经丛中ntsr1的表达,光镜下观察并统计其阳性表达情况,并与he染色结果进行对比。同时应用计算机图像分析软件(image-proplus6.0)计算ntsr1在实验组及对照组各段肠壁肌间神经丛中免疫组化阳性染色的平均光密度(mod)并将结果进行定量分析,将所得数据用spss13.0统计软件进行处理分析,判定差别是否存在意义。结果1.he染色:狭窄段肌间神经丛中未见神经节细胞,神经纤维较扩张段明显增粗、排列紊乱。从狭窄段到正常段肠壁肌间神经丛中,神经节细胞的分布逐渐增多,变性神经纤维的分布逐渐减少。2.在hscr实验组正常段及对照组肌间神经丛中,可见ntsr1强阳性表达的神经纤维;在扩张段,强阳性表达的神经纤维较实验组正常段和对照组有所减少;在移行段,可见散在弱阳性到中等强度阳性表达的神经纤维;在狭窄段,可见少量弱阳性表达的神经纤维,肌间神经丛总体表达呈弱阳性或阴性。3.ntsr1在hscr各段肠壁平滑肌中呈弱阳性到中等强度阳性表达,其中环形肌的总体表达强度高于纵形肌。4.定量分析:ntsr1在hscr实验组狭窄段、移行段、扩张段、正常段及对照组肌间神经丛中阳性表达的平均光密度分别为8.39±2.68、11.76±6.79、17.14±12.26、21.92±14.68、23.45±19.82,经统计软件分析,与移行段、扩张段、正常段及对照组肠壁肌间神经丛相比,狭窄段平均光密度明显降低,差异有统计学意义(P0.05);移行段与扩张段、移行段与正常段、移行段与对照组、扩张段与正常段、扩张段与对照组、正常段与对照组各间的平均光密度相比,没有明显差异(P0.05)。结论1.狭窄段肠壁肌间神经丛中NTSR1表达明显降低,其表达异常可能是导致HSCR肠蠕动功能障碍的重要原因之一。2.免疫组化显示NTSR1在HSCR的狭窄段肠壁肌间神经丛大多表达阴性,少部分表达呈弱阳性,这对活检诊断HSCR有一定的帮助。NTSR1有望作为筛选诊断HSCR的一种敏感有效的神经标志物。
[Abstract]:The purpose of Hirschsprung's disease (Hirschsprung 's disease, HSCR) is also called "intestinal aganglionosis" by genetic factors, environment as a result of the enteric nervous system (Enteric nervous system, ENS) early occurrence of multiple mutations or a congenital intestinal disease of abnormal neural crest cells the expression. The basic pathophysiological changes of distal colonic submucosal plexus (Submucosal plexuses) and myenteric plexus (Myenteric plexuses) in the complete or partial lack of intestinal ganglion cells (Intestinal ganglion cells, IGCS), the nerve fibers arranged disorderly, wavy, caused by the continued loss of normal colonic spasm. Promote peristalsis. As the stool through the difficult, long-term deposition in spastic intestinal segments, proximal colon compensatory expansion, thickening, formation of giant colon.ENS is a complex and active gastrointestinal motility and sense Feel the nervous system composed of intestinal myenteric and submucosal plexus, close contact between the two, on gastrointestinal motility, secretion function with independent regulation of.HSCR ENS early gene mutation or loss of function. The results of neurotensin (neurotensin, NT) is a neurotransmitter or neuromodulator secretion open type by intestinal has special endocrine function of N cells, the transcription factor belongs to ligand dependent, in the central nervous system (Central nervous system, CNS) and the cardiovascular, respiratory, digestive, endocrine, play an important role in immune function and other peripheral systems. Neurotensin receptor 1 (Neurotensin receptor 1, NTSR1) is one of the 7 transmembrane domain G protein coupled receptors in human colon and the highest affinity ligand NT.NT mediated by G protein coupled receptor NTSR1 activates the corresponding letter Signaling pathway has a downward signal transmission to gastrointestinal tract smooth muscle effect site, thereby enhancing colonic propulsive movement, so that stool drain.NTSR1 in ENS on intestinal motility plays an important role. In our previous study, using gene chip technology to detect the HSCR expression of a large number of genes, including ntsr1 gene the expression was down regulated, and verified by RT-PCR. Access to relevant literature, the relationship between domestic and foreign nt-ntsr1 interactions and the pathogenesis of HSCR. There is less research studies have found that ntsr1 nerve in normal human colon tissue of submucosal and myenteric plexus have different degree of positive expression, ntsr1 and HSCR in different intestinal segments expression and the abnormal expression is not clear. Therefore, this study selected the ntsr1 gene from the previous research results, by immunohistochemical staining of ntsr1 HSCR in the wall of intestine The expression of the myenteric plexus, and to investigate the possible relationship between ntsr1 and the pathogenesis of HSCR. Methods HSCR patients from January 2010 to September 2014 in Guangdong Provincial Maternity and Child Care Center surgical specimens of pediatric surgery through the anus megacolon radical operation in the treatment of 42 patients as the experimental group, the experimental group was divided into stenosis segment, transitional segment, expansion section, normal section four for the first time groups. All patients were underwent surgical treatment, were sporadic cases, with no other major organs or congenital malformation syndrome, near relatives were no HSCR cases. All patients according to the clinical manifestations of preoperative barium enema examination, pathology and postoperative pathology, two physician HE staining combined the results confirmed diagnosis and intraoperative frozen section. The selected cases with normal distribution. The principle of selection is similar to the experimental group age such as congenital anal and colonic perforation, dolichasigmoid Such as resection of non HSCR in children with normal colon specimens in 20 cases as control group, control HSCR cases and other intestinal neuronal dysplasia disease group family. Experimental groups and control groups were statistical results and he staining under optical microscope. Immunohistochemical staining was used to detect 42 cases of experimental group were stenosis and the transitional segment, the expansion section, the normal segment and 20 cases of normal control group were intestinal myenteric plexus of the expression of ntsr1 was observed under light microscope and statistics of its expression, and the results were compared with HE staining. At the same time, the application of computer image analysis software (image-proplus6.0) to calculate ntsr1 in the experimental group and control group the wall of intestine myenteric plexus of immunohistochemical staining of the average optical density (MOD) and the results of quantitative analysis of the data using SPSS13.0 statistical software to analysis, to determine whether there is difference Results: 1.he staining. No stenosis of the myenteric plexus of the ganglion cells and nerve fibers with Duan Mingxian expansion thickening, arranged in disorder. From the stricture to normal segments of intestinal myenteric plexus, distribution of ganglion cells gradually increased, the distribution of degeneration of neural fibers decreased.2. in HSCR experimental group and normal control group of myenteric plexus, showed strong positive expression of ntsr1 in nerve fiber; expansion section, strong expression of nerve fibers than in the experimental group and normal control group decreased; in the transitional period, scattered in the weak to moderate positive nerve fibers positive expression; in the narrow segment, a small amount of weak positive expression of nerve fibers and myenteric plexus of the overall negative or weakly positive expression of.3.ntsr1 was weakly positive to moderate positive expression of HSCR in the wall of intestine smooth muscle, the overall circular muscle expression of high strength in longitudinal muscle Quantitative analysis of.4.: ntsr1 in HSCR experimental group stenosis segment, transitional segment, expansion, and normal control group in myenteric plexus of the positive expression of the average density was 8.39 + 2.68,11.76 + 6.79,17.14 + 12.26,21.92 + 14.68,23.45 + 19.82, by statistical analysis software, the expansion section and the transitional segment, and compared to the normal section and the control group of myenteric plexus, stenosis of the average optical density decreased significantly, the difference was statistically significant (P0.05); transitional section and expansion section, transitional segments and normal segments, the transitional segment with the control group, and the normal period of expansion, expansion section and the control group, compared with the normal section the average optical density of the control group, no significant difference (P0.05). Conclusion: 1. stenosis of myenteric plexus of the expression of NTSR1 was significantly decreased, the abnormal expression may be one of the important causes of.2. immunohistochemistry showed HSCR motility dysfunction in NTSR1 HSCR intestinal stricture Most of the intermuscular plexus is negative, and a few of them are weakly positive, which is helpful for biopsy diagnosis of HSCR..NTSR1 is expected to be a sensitive and effective neural marker for screening and diagnosing HSCR.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R726.5

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