本文關(guān)鍵詞：神經(jīng)降壓素受體1在先天性巨結(jié)腸癥腸壁肌間神經(jīng)叢的表達(dá)及意義　出處：《廣州醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 先天性巨結(jié)腸 NTSR1 免疫組化 肌間神經(jīng)叢 腸神經(jīng)系統(tǒng)

【摘要】：目的先天性巨結(jié)腸癥(Hirschsprung’s disease,HSCR)又稱“腸壁無神經(jīng)節(jié)細(xì)胞癥”,是由遺傳、環(huán)境等多種因素共同作用導(dǎo)致腸神經(jīng)系統(tǒng)(Enteric nervous system,ENS)早期發(fā)生多基因突變或表達(dá)異常的神經(jīng)嵴細(xì)胞源性的一種先天性腸道疾病。其基本病理生理改變?yōu)檫h(yuǎn)端病變結(jié)腸黏膜下神經(jīng)叢(Submucosal plexuses)和肌間神經(jīng)叢(Myenteric plexuses)內(nèi)完全或部分缺乏腸神經(jīng)節(jié)細(xì)胞(Intestinal ganglion cells,IGCS),致使神經(jīng)纖維排列紊亂,呈波浪狀,造成結(jié)腸持續(xù)痙攣,失去正常的推進(jìn)蠕動(dòng)功能。由于糞便通過困難,長(zhǎng)期淤積于痙攣腸段,近端結(jié)腸代償性擴(kuò)張、增厚,形成巨結(jié)腸。ENS是一個(gè)復(fù)雜而積極的胃腸道動(dòng)力和感覺神經(jīng)系統(tǒng),由腸道肌間和黏膜下神經(jīng)叢共同構(gòu)成,兩者之間緊密聯(lián)系,對(duì)胃腸道的運(yùn)動(dòng)、分泌功能具有獨(dú)立的調(diào)節(jié)作用。HSCR是ENS早期發(fā)生基因突變或功能喪失的結(jié)果。神經(jīng)降壓素(neurotensin,NT)是由腸道具有特殊內(nèi)分泌功能的開放型的N細(xì)胞分泌的一種神經(jīng)遞質(zhì)或調(diào)質(zhì),屬于配體依賴性的轉(zhuǎn)錄因子,在中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)以及心血管、呼吸、消化、內(nèi)分泌、免疫等多種外周系統(tǒng)中存在并發(fā)揮重要的功能調(diào)節(jié)作用。神經(jīng)降壓素受體1(Neurotensin receptor 1,NTSR1)是一種具有7個(gè)跨膜結(jié)構(gòu)域的G蛋白偶聯(lián)受體,在人體結(jié)腸中與NT配基親和力最高。NT通過介導(dǎo)G蛋白偶聯(lián)受體NTSR1激活相應(yīng)的信號(hào)通路,產(chǎn)生向下的信號(hào)傳遞,到達(dá)胃腸道平滑肌等效應(yīng)部位,從而增強(qiáng)結(jié)腸的推進(jìn)運(yùn)動(dòng),使糞便排空。NTSR1在ENS中對(duì)腸道動(dòng)力具有十分重要的作用。在我們的前期研究中,利用基因芯片技術(shù)檢測(cè)出大量的HSCR差異性表達(dá)基因,其中ntsr1基因表達(dá)明顯下調(diào),并經(jīng)rt-pcr驗(yàn)證。查閱相關(guān)文獻(xiàn),目前國(guó)內(nèi)外在nt-ntsr1的相互作用與hscr發(fā)病的相關(guān)關(guān)系方面研究較少。有研究已發(fā)現(xiàn),ntsr1在人體正常結(jié)腸組織黏膜下和肌間神經(jīng)叢存在不同程度的陽(yáng)性表達(dá),而ntsr1在hscr各腸段是否表達(dá)并存在表達(dá)的異常尚不清楚。因此,本研究從前期研究結(jié)果中選取ntsr1基因,采用免疫組織化學(xué)染色法觀察ntsr1在hscr各段腸壁肌間神經(jīng)叢的表達(dá)情況,從而初步探討ntsr1與hscr發(fā)病的可能關(guān)系。方法選取2010年1月至2014年9月在廣東省婦幼保健院小兒外科經(jīng)肛門巨結(jié)腸根治術(shù)治療的hscr患兒手術(shù)標(biāo)本42例作為實(shí)驗(yàn)組,實(shí)驗(yàn)組分為狹窄段、移行段、擴(kuò)張段、正常段四個(gè)組別。所有患兒均為首次接受手術(shù)治療,均為散發(fā)病例,無合并其他主要臟器的先天性畸形或綜合癥,近系親屬均無hscr病例。所有患兒均根據(jù)術(shù)前臨床表現(xiàn)、鋇劑灌腸造影等檢查,以及術(shù)中快速冰凍病理及術(shù)后病理科醫(yī)師二次he染色診斷結(jié)果綜合確診。所選取病例符合正態(tài)分布的原則。選取與實(shí)驗(yàn)組年齡相近的如先天性無肛門、結(jié)腸穿孔、乙狀結(jié)腸冗長(zhǎng)癥等非hscr患兒手術(shù)切除的正常結(jié)腸標(biāo)本20例作為對(duì)照組,對(duì)照組家族中無hscr病例及其它腸神經(jīng)發(fā)育異常性疾病。在光學(xué)顯微鏡下觀察實(shí)驗(yàn)組與對(duì)照組病例he染色的結(jié)果并統(tǒng)計(jì)。應(yīng)用免疫組織化學(xué)染色法檢測(cè)42例實(shí)驗(yàn)組標(biāo)本狹窄段、移行段、擴(kuò)張段、正常段及20例正常對(duì)照組標(biāo)本腸壁肌間神經(jīng)叢中ntsr1的表達(dá),光鏡下觀察并統(tǒng)計(jì)其陽(yáng)性表達(dá)情況,并與he染色結(jié)果進(jìn)行對(duì)比。同時(shí)應(yīng)用計(jì)算機(jī)圖像分析軟件(image-proplus6.0)計(jì)算ntsr1在實(shí)驗(yàn)組及對(duì)照組各段腸壁肌間神經(jīng)叢中免疫組化陽(yáng)性染色的平均光密度(mod)并將結(jié)果進(jìn)行定量分析,將所得數(shù)據(jù)用spss13.0統(tǒng)計(jì)軟件進(jìn)行處理分析,判定差別是否存在意義。結(jié)果1.he染色:狹窄段肌間神經(jīng)叢中未見神經(jīng)節(jié)細(xì)胞,神經(jīng)纖維較擴(kuò)張段明顯增粗、排列紊亂。從狹窄段到正常段腸壁肌間神經(jīng)叢中,神經(jīng)節(jié)細(xì)胞的分布逐漸增多,變性神經(jīng)纖維的分布逐漸減少。2.在hscr實(shí)驗(yàn)組正常段及對(duì)照組肌間神經(jīng)叢中,可見ntsr1強(qiáng)陽(yáng)性表達(dá)的神經(jīng)纖維;在擴(kuò)張段,強(qiáng)陽(yáng)性表達(dá)的神經(jīng)纖維較實(shí)驗(yàn)組正常段和對(duì)照組有所減少;在移行段,可見散在弱陽(yáng)性到中等強(qiáng)度陽(yáng)性表達(dá)的神經(jīng)纖維;在狹窄段,可見少量弱陽(yáng)性表達(dá)的神經(jīng)纖維,肌間神經(jīng)叢總體表達(dá)呈弱陽(yáng)性或陰性。3.ntsr1在hscr各段腸壁平滑肌中呈弱陽(yáng)性到中等強(qiáng)度陽(yáng)性表達(dá),其中環(huán)形肌的總體表達(dá)強(qiáng)度高于縱形肌。4.定量分析:ntsr1在hscr實(shí)驗(yàn)組狹窄段、移行段、擴(kuò)張段、正常段及對(duì)照組肌間神經(jīng)叢中陽(yáng)性表達(dá)的平均光密度分別為8.39±2.68、11.76±6.79、17.14±12.26、21.92±14.68、23.45±19.82,經(jīng)統(tǒng)計(jì)軟件分析,與移行段、擴(kuò)張段、正常段及對(duì)照組腸壁肌間神經(jīng)叢相比,狹窄段平均光密度明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);移行段與擴(kuò)張段、移行段與正常段、移行段與對(duì)照組、擴(kuò)張段與正常段、擴(kuò)張段與對(duì)照組、正常段與對(duì)照組各間的平均光密度相比,沒有明顯差異(P0.05)。結(jié)論1.狹窄段腸壁肌間神經(jīng)叢中NTSR1表達(dá)明顯降低,其表達(dá)異�？赡苁菍�(dǎo)致HSCR腸蠕動(dòng)功能障礙的重要原因之一。2.免疫組化顯示NTSR1在HSCR的狹窄段腸壁肌間神經(jīng)叢大多表達(dá)陰性,少部分表達(dá)呈弱陽(yáng)性,這對(duì)活檢診斷HSCR有一定的幫助。NTSR1有望作為篩選診斷HSCR的一種敏感有效的神經(jīng)標(biāo)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.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R726.5

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