本文選題：炎癥性腸病 + 腸壁纖維化��； 參考：《南昌大學(xué)》2014年碩士論文

【摘要】：研究背景： 炎癥性腸�。╥nflammatory bowel disease,IBD）包括潰瘍性結(jié)腸炎(ulcerativecolitis,UC)及克羅恩病(Crohn disease,CD)，為腸道的慢性非特異性炎性疾病，其病因和發(fā)病機制尚不確切，近年來，其發(fā)病率持續(xù)增高，引起了人們高度的重視。長期的慢性炎癥刺激能導(dǎo)致腸壁纖維化，且克羅恩病的病變累及范圍廣，為腸壁全層性炎癥，導(dǎo)致大量細胞外基質(zhì)（extracellular matrix,ECM）異常沉淀引起腸壁各解剖層發(fā)生纖維化，易產(chǎn)生瘢痕性狹窄從而導(dǎo)致腸梗阻。目前，對于慢性腸壁纖維化的治療，傳統(tǒng)的治療方法及外科手術(shù)治療效果均不理想，預(yù)后不佳，尚缺乏十分有效的治療手段。國內(nèi)外研究發(fā)現(xiàn)，ECM的沉淀以膠原蛋白的沉積為主，賴氨酸羥化酶（lysyl hydroxylase,LH）在膠原蛋白的形成及穩(wěn)定中起著重要作用，其中尤以LH2作用最為關(guān)鍵，而PLOD(procollage-lysine,2-oxiglutarate,5-dioxygerase)基因為LH的編碼基因，，因此，PLOD2有望成為預(yù)防或治療慢性腸纖維化的研究新方向。 目的： 前期研究表明，應(yīng)用三硝基苯磺酸（trinitro-benzene-sulfonic acid,TNBS）灌腸可成功誘導(dǎo)BALB/C小鼠慢性腸纖維化模型的建立，在此基礎(chǔ)上，我們合成PLOD2反義寡核苷酸，以此干預(yù)灌腸來探討PLOD2反義寡核苷酸對BALB/C小鼠慢性腸壁纖維化的影響及作用機制。通過比較實驗各組間小鼠的疾病活動指數(shù)（DAI），觀察結(jié)腸組織的病理變化和膠原纖維的增生程度，比較分析TNF-α、PLOD2、Col-ⅢmRNA的表達水平及Col-Ⅲ蛋白的表達水平，進而分析PLOD2反義寡核苷酸在TNBS誘導(dǎo)小鼠慢性腸壁纖維化中的作用，探討其作用機制，并為PLOD2反義寡核苷酸治療慢性腸壁纖維化及開發(fā)相關(guān)基因治療藥物提供一定的實驗及理論依據(jù)。 實驗方法： 隨機將40只體重為20-25g、6-8周齡的雌性BALB/C小鼠分為4組，每組10只，分別為生理鹽水空白對照組（空白對照組），TNBS模型組（TNBS組），PLOD2錯義寡核苷酸陰性對照組（MSODN組）及PLOD2反義寡核苷酸治療組（ASODN組）。對BALB/C小鼠采用灌腸的方式建立動物模型，空白對照組每周給予生理鹽水100ul灌腸后24小時再次給予生理鹽水100ul灌腸；TNBS模型組每周先給予2mg TNBS/50%乙醇溶液100u1灌腸，24小時后給予生理鹽水100ul灌腸；PLOD2錯義寡核苷酸陰性對照組每周先給予2mg TNBS/50%乙醇溶液100u1灌腸，24小時后給予PLOD2錯義寡核苷酸100ul灌腸；PLOD2反義寡核苷酸治療組每周先給予2mg TNBS/50%乙醇溶液100u1灌腸，24小時后給予PLOD2反義寡核苷酸100ul灌腸；每組均連續(xù)灌腸6周，于最后1次灌腸后1周采取頸椎脫臼法處死小鼠并取結(jié)腸組織。采集的結(jié)腸組織分別進行如下處理：即HE染色評估結(jié)腸組織炎癥程度；VG染色評估結(jié)腸組織纖維化程度；RT-PCR檢測結(jié)腸組織中TNF-α、PLOD2、COL-III的mRNA的表達；免疫組化檢測結(jié)腸組織中COL-III蛋白的表達水平。 結(jié)果： 1.TNBS組、MSODN組及ASODN組小鼠每次灌腸后出現(xiàn)不同程度的癥狀，如少食，少動，身體蜷縮，稀便，大便潛血陽性甚至肉眼血便，皮毛光澤度下降等，第3-4天后上述癥狀逐漸減輕。小鼠的體重大部分在第2-4天有不同程度的下降，部分第4天后體重較前有所增加，甚至超過灌腸前體重。整個實驗期間，癥狀以前3周最重，第4周后癥狀相對減輕。三組小鼠均有個別小鼠死亡，死亡時間主要在前3周。空白對照組灌腸后進食及活動情況無明顯改變，無小鼠死亡。TNBS組、MSODN組及ASODN組小鼠DAI評分均高于空白對照組（P＜0.05）；TNBS組、MSODN組及ASODN組組間相比較無統(tǒng)計學(xué)意義（P0.05）。 2.肉眼觀察各組小鼠結(jié)腸組織的大體表現(xiàn)，可見TNBS組、MSODN組及ASODN組有不同程度的充血、水腫、粘連等，剖開后腸壁有不同程度的糜爛、潰瘍，有的部位出現(xiàn)管壁增厚、狹窄、僵硬變形等表現(xiàn)，空白對照組小鼠結(jié)腸組織則無上述改變。HE染色鏡下觀察空白組小鼠結(jié)腸組織無明顯炎癥表現(xiàn)，TNBS組、MSODN組及ASODN組都可見不同程度的上皮細胞、腺體及隱窩的破壞，杯狀細胞減少，以淋巴細胞及單核細胞為主的炎癥浸潤，可有淋巴濾泡增生，部分結(jié)腸組織可出現(xiàn)粘膜層潰瘍，部分甚至達粘膜肌層。TNBS組、MSODN組及ASODN組小鼠炎癥評分均高于空白對照組（P＜0.05）；而這三組間相比較無統(tǒng)計學(xué)意義（P0.05）。VG染色鏡下可見空白對照組小鼠結(jié)腸組織無明顯纖維化表現(xiàn)，而TNBS組及MSODN組中小鼠結(jié)腸組織中可見大量膠原蛋白沉積，固有肌層可有不同程度的增厚，部分甚至可見纖維分隔。ASODN組中小鼠結(jié)腸組織的膠原蛋白沉積及固有肌層增厚程度較TNBS組及MSODN組輕。TNBS組、MSODN組及ASODN組小鼠纖維化評分均高于空白對照組（P＜0.05）；TNBS組及MSODN組組間相比較無統(tǒng)計學(xué)意義（P0.05）；而ASODN組評分低于TNBS組及MSODN組（P＜0.05）。 3.TNBS組、MSODN組中PLOD2mRNA的表達高于空白對照組及ASODN組（P＜0.05），ASODN組高于空白組（P＜0.05），TNBS組及MSODN組間比較無統(tǒng)計學(xué)意義（P＞0.05）。對于TNF-αmRNA及Col-IIImRNA的表達，TNBS組、MSODN組及ASODN組的表達均高于空白對照組（P＜0.05），但這三組間比較無統(tǒng)計學(xué)意義（P＞0.05）。 4.TNBS組、MSODN組及ASODN組Col-III蛋白的表達較空白對照組高（P＜0.05），但ASODN組比TNBS組、MSODN組表達低（P＜0.05），而TNBS組及MSODN組間比較無統(tǒng)計學(xué)意義（P＞0.05）。 結(jié)論： 應(yīng)用PLOD2ASODN治療TNBS/50%EtOH誘導(dǎo)的小鼠慢性腸纖維化，其通過抑制PLOD2的表達，降低LH2的活性，從而減少III型膠原蛋白的合成，降低腸壁纖維化的程度。
[Abstract]:Research background:
Inflammatory bowel disease (IBD), which includes ulcerative colitis (ulcerativecolitis, UC) and Crohn's disease (Crohn disease, CD), is a chronic nonspecific inflammatory disease of the intestines. Its etiology and pathogenesis are still uncertain. In recent years, the incidence of the disease has been increasing, and people have paid great attention to the chronic inflammatory disease. Irritable energy leads to intestinal wall fibrosis, and the lesions of Crohn's disease are involved in a wide range, as a whole layer of intestinal inflammation, resulting in a large number of extracellular matrix (extracellular matrix, ECM) abnormal precipitations that cause fibrosis in the anatomic layers of the intestinal wall, causing scar stricture and causing intestinal obstruction. The results of the treatment and the surgical treatment are not ideal, and the prognosis is not good. There is still a lack of effective treatment. It is found that the precipitation of ECM is mainly deposited by collagen, and lysyl hydroxylase (LH) plays an important role in the formation and stability of collagen, especially the role of LH2 is the most important. The gene of PLOD (procollage-lysine, 2-oxiglutarate, 5-dioxygerase) is the encoding gene of LH. Therefore, PLOD2 is expected to be a new research direction for the prevention or treatment of chronic intestinal fibrosis.
Objective:
Previous studies have shown that Trinitro-benzene-sulfonic acid (Trinitro-benzene-sulfonic acid, TNBS) enema can successfully induce chronic intestinal fibrosis in BALB/C mice. On this basis, we synthesize PLOD2 antisense oligodeoxynucleotides to investigate the effect of PLOD2 antisense oligodeoxynucleotides on the chronic intestinal fibrosis in BALB/C mice and the effect of PLOD2 antisense oligodeoxynucleotides on the chronic intestinal fibrosis in BALB/C mice. By comparing the disease activity index (DAI) of mice in the experimental groups, the pathological changes of colonic tissue and the degree of proliferation of collagen fibers were observed. The expression level of TNF- alpha, PLOD2, Col- III mRNA and the expression level of Col- III protein were compared and analyzed, and then the PLOD2 antisense oligodeoxynucleotides were analyzed in the chronic intestinal fibrosis induced by TNBS in mice. The role of PLOD2 antisense oligodeoxynucleotides for the treatment of chronic intestinal fibrosis and the development of related gene therapy drugs for the development of antisense oligodeoxynucleotides provide some experimental and theoretical basis.
Experimental methods:
The female BALB/C mice of 40 20-25g and 6-8 weeks old were randomly divided into 4 groups, 10 in each group, respectively, the blank control group (blank control group), the TNBS model group (group TNBS), the PLOD2 missense oligonucleotide negative control group (group MSODN) and the PLOD2 antisense oligoside acid treatment group (ASODN group). The enema method was established in the BALB/C mice. In the blank control group, the blank control group was given the saline 100ul enema again after 24 hours of saline 100ul enema every week; the TNBS model group gave 2mg TNBS/50% ethanol solution 100u1 enema first, and then given the saline 100ul enema after 24 hours, and the PLOD2 missense oligonucleotide negative control group gave 2mg TNBS/50% ethanol solution 100u1 every week. The enema was given after 24 hours of PLOD2 missense oligonucleotide 100ul enema; PLOD2 antisense oligodeoxynucleotides were given 100u1 enema by 2mg TNBS/50% ethanol solution and PLOD2 antisense oligodeoxynucleotides 100ul enema 24 hours later; each group was consecutively enema for 6 weeks, and the cervical dislocations were executed and colonic group was taken 1 weeks after the last 1 enema. The colonic tissues collected were treated as following: HE staining was used to assess the degree of inflammation in colon tissue; VG staining was used to assess the degree of fibrosis in colon tissue; RT-PCR was used to detect the expression of mRNA in the colon of TNF- a, PLOD2, COL-III; and immunohistochemistry was used to detect the expression level of COL-III egg white in colon tissue.
Result錛

本文編號：2019480