本文選題：Cajal間質(zhì)細(xì)胞 + 炎癥細(xì)胞因子��； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 本實(shí)驗(yàn)主要利用旋毛蟲感染C57BL\6小鼠建立PI-IBS動(dòng)物模型，對(duì)該模型的內(nèi)臟敏感性和腸道動(dòng)力進(jìn)行評(píng)價(jià)，檢測(cè)各腸段中c-kit蛋白、IL-1β、IL-10、IL-17和IFN-γ表達(dá)，分析c-kit蛋白與炎癥因子的關(guān)系，初步探討腸道ICC與炎癥細(xì)胞因子改變?cè)赑I-IBS發(fā)病機(jī)制中的作用。 方法 1. PI-IBS小鼠模型的建立：34只雌性C57BL/6小鼠隨機(jī)分為PI-IBS組（17只）和對(duì)照組（17只），PI-IBS組用0.2ml含400條旋毛蟲幼蟲的生理鹽水懸液灌胃感染，對(duì)照組給予等量的生理鹽水。 2.蘇木精-伊紅染色（hematoxylin-eosin staining, HE staining）：感染后第14天、28天和56天小鼠的十二指腸、空腸、末端回腸和近端結(jié)腸組織做病理學(xué)檢查，觀察炎癥改變。 3.內(nèi)臟敏感性和腸道動(dòng)力評(píng)價(jià):對(duì)感染第56天小鼠進(jìn)行結(jié)直腸擴(kuò)張（colorectal distention，CRD），觀察小鼠的腹壁撤退反射（abdominalwithdrawal reflex，AWR）并進(jìn)行評(píng)分；檢測(cè)小鼠腸道傳輸時(shí)間（intestinetransmit time，ITT）并統(tǒng)計(jì)每2小時(shí)的糞便粒數(shù)、Bristol評(píng)分、濕/干重和含水百分?jǐn)?shù)。 4.炎癥細(xì)胞因子和c-kit蛋白表達(dá)的檢測(cè):用酶聯(lián)免疫吸附測(cè)定（enzyme linked immunosobrent assay，ELISA）檢測(cè)不同腸段組織蛋白勻漿液中IL-1β、IL-10、IL-17和IFN-γ的表達(dá)水平；用免疫組織化學(xué)染色檢測(cè)不同腸道組織中c-kit蛋白表達(dá)的部位，并對(duì)c-kit蛋白染色程度和c-kit蛋白陽(yáng)性細(xì)胞比例進(jìn)行綜合評(píng)分；用western blotting法對(duì)不同腸道組織的蛋白勻漿液中c-kit蛋白進(jìn)行半定量分析。 5.炎癥因子和c-kit信使核糖核酸(message ribonucleic acid, mRNA)檢測(cè)：通過(guò)反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)（reverse transcription polymerasechain reaction, RT-PCR）檢測(cè)各腸道組織中c-kitmRNA、IL-1βmRNA、IL-10mRNA、IL-17mRNA和IFN-γmRNA的表達(dá)。 結(jié)果 1. PI-IBS組小鼠的十二指腸、空腸、末端回腸和近端結(jié)腸組織在旋毛蟲感染后第14天存在顯著的炎癥細(xì)胞浸潤(rùn)和間質(zhì)水腫，第14天到28天炎癥和水腫逐漸減輕，至第56天基本恢復(fù)正常。 2.當(dāng)CRD的空氣容量為0.35ml和0.5ml時(shí)，PI-IBS組小鼠的AWR評(píng)分明顯高于對(duì)照組小鼠，，且差異具有統(tǒng)計(jì)學(xué)意義（P0.01）。與對(duì)照組小鼠比較，PI-IBS組的ITT明顯短于對(duì)照組（P0.01）；每2小時(shí)的糞便粒數(shù)、Bristol評(píng)分、濕重和含水百分?jǐn)?shù)均高于對(duì)照組（P0.05）,兩組小鼠糞便干重差別無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 3.與對(duì)照組小鼠比較，PI-IBS小鼠的IFN-γ和IL-17在十二指腸和回腸組織中表達(dá)升高(P0.05);IL-10在空腸、回腸和結(jié)腸組織中的表達(dá)均降低（P0.05），c-kit在十二指腸、空腸、回腸和結(jié)腸組織中的表達(dá)均升高（P0.05），IL-1β在各腸段組織中表達(dá)的差異無(wú)統(tǒng)計(jì)學(xué)意義。 4.免疫組織化學(xué)染色提示c-kit陽(yáng)性信號(hào)主要分布在腸道組織的固有層和肌肉層。PI-IBS小鼠各腸段組織染色程度和c-kit陽(yáng)性細(xì)胞比例的綜合評(píng)分明顯高于對(duì)照小鼠（P0.05）。 結(jié)論 1.旋毛蟲感染C57BL\6小鼠能較好的模擬PI-IBS內(nèi)臟高敏感性和腸道動(dòng)力改變，可作為PI-IBS動(dòng)物模型。 2. PI-IBS小鼠腸道組織的ICC數(shù)量和c-kit蛋白表達(dá)量的改變可能與內(nèi)臟敏感性和腸道動(dòng)力改變相關(guān)。 3. PI-IBS小鼠腸道組織中炎癥因子的改變可能與ICC改變有關(guān)，參與PI-IBS的發(fā)病機(jī)制。
[Abstract]:Purpose

In this experiment , we established a PI - IBS animal model by using Trichina spiralis . The visceral sensitivity and intestinal motility of the model were evaluated . The expression of c - kit protein , IL - 1尾 , IL - 10 , IL - 17 and IFN - 緯 in various intestinal segments was detected . The relationship between c - kit protein and inflammatory factor was analyzed .

method

1 . Establishment of the model of PI - IBS mice : 34 female C57BL / 6 mice were randomly divided into PI - IBS group ( 17 rats ) and control group ( 17 rats ) . The PI - IBS group was infected with 0.2 ml physiological saline suspension containing 400 spiralis larvae , and the control group gave equivalent normal saline .

2 . Hematoma - eosin staining ( HE staining ) : The duodenum , jejunum , distal ileum and proximal colon tissues of 14 , 28 , and 56 days post - infection were examined by pathology to observe the changes of inflammation .

3 . Visceral sensitivity and intestinal motility evaluation : colorectal distension ( CRD ) was performed on 56 day - infected mice , and the abdominal wall withdrawal reflex ( AWR ) of mice was observed and scored ;
Mice intestinal transit time ( ITT ) was tested and the number of fecal pellets , Bristol score , wet / dry weight , and water content per 2 hours were counted .

4 . Detection of inflammatory cytokines and c - kit protein expression : The levels of IL - 1尾 , IL - 10 , IL - 17 and IFN - 緯 were detected by enzyme linked immunoadsorption assay ( ELISA ) .
the expression of c - kit protein in different intestinal tissues was detected by immunohistochemical staining , and the degree of c - kit protein staining and the proportion of c - kit protein positive cells were scored ;
The c - kit protein was semi - quantitatively analyzed by western blotting .

5.鐐庣棁鍥犲瓙鍜宑-kit淇′嬌鏍哥硸鏍擱吀(message ribonucleic acid, mRNA)媯

本文編號(hào)：1924069