本文選題：急性胰腺炎 + CUEDC2　； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：急性胰腺炎(acutepancreatitis,AP)是臨床常見急腹癥,具有起病急、病情重、變化快等特點(diǎn),除了胰腺本身病變之外,還可能引起胰腺外器官和組織的損傷,甚至最終誘發(fā)全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome,SIRS)、多器官功能障礙綜合征(multiple-organ dysfunction syndrome,MODS)等而導(dǎo)致死亡。盡管在過去的幾十年里急性胰腺炎的研究取得明顯進(jìn)展,但是其發(fā)病機(jī)制仍較復(fù)雜。急性胰腺炎(acute pancreatitis,AP)無論病因如何,損傷的胰腺會(huì)引起胰酶的自身消化,同時(shí)作為炎癥刺激物刺激單核巨噬細(xì)胞及胰腺腺泡細(xì)胞釋放炎癥介質(zhì)和細(xì)胞因子,觸發(fā)炎癥介質(zhì)形成瀑布樣級聯(lián)反應(yīng),最終會(huì)導(dǎo)致局部或全身炎癥反應(yīng)。CUEDC2(CUE domain-containing 2)是一種含有CUE結(jié)構(gòu)域的多功能蛋白質(zhì),廣泛在人類組織和器官中表達(dá)。大量研究表明,CUEDC2可以作用于多種細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)通路,參與許多細(xì)胞活動(dòng),如細(xì)胞周期的調(diào)節(jié),生長信號因子的轉(zhuǎn)導(dǎo)、腫瘤的發(fā)生發(fā)展及炎癥反應(yīng)。目前研究發(fā)現(xiàn)CUEDC2不僅與腫瘤的發(fā)生發(fā)展密切相關(guān),如乳腺癌、結(jié)腸炎相關(guān)性腫瘤、肺癌等,還可以參與炎癥反應(yīng),如結(jié)腸炎等。關(guān)于CUEDC2與急性胰腺炎的炎癥反應(yīng)中的關(guān)系尚未有研究報(bào)道。本研究擬初步探討CUEDC2在急性胰腺炎中的作用。目的:本研究通過雨蛙素誘導(dǎo)AR42J細(xì)胞建立急性胰腺炎細(xì)胞模型,并利用質(zhì)粒轉(zhuǎn)染技術(shù)過表達(dá)CUEDC2。監(jiān)測細(xì)胞培養(yǎng)液中淀粉酶(amylase,AMY)、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細(xì)胞介素-6(interleukin-6,IL-6)的水平,初步探討CUEDC2對急性胰腺炎腺泡細(xì)胞TNF-α、IL-6的影響。方法:1應(yīng)用雨蛙素誘導(dǎo)AR42J細(xì)胞建立急性胰腺炎細(xì)胞模型。2獲得過表達(dá)CUEDC2的AR42J細(xì)胞:采用脂質(zhì)體轉(zhuǎn)染法將CUEDC2質(zhì)粒轉(zhuǎn)入AR42J細(xì)胞中,應(yīng)用熒光顯微鏡查看轉(zhuǎn)染效率。3實(shí)驗(yàn)分組:AR42J細(xì)胞分為4組:陰性質(zhì)粒對照組(Neg-Control組)、陰性質(zhì)粒雨蛙素組(Neg-CAE組)、CUEDC2質(zhì)粒對照組(CUEDC2-Control組)、CUEDC2質(zhì)粒雨蛙素組(CUEDC2-CAE組)。具體分組方法如下:陰性質(zhì)粒組:應(yīng)用陰性質(zhì)粒轉(zhuǎn)染AR42J細(xì)胞。Neg-CAE組給予10-7 mol/L雨蛙素,Neg-Control組給予等體積完全培養(yǎng)基,再將兩組分為3個(gè)亞組:4h、8h、24h。CUEDC2質(zhì)粒組:應(yīng)用CUEDC2質(zhì)粒轉(zhuǎn)染AR42J細(xì)胞。CUEDC2-CAE組給予10-7mol/L雨蛙素 CUEDC2-Control組給予等體積完全培養(yǎng)基,再將兩組分為3個(gè)亞組:4h、8 h、24h。4 CUEDC2 測定:PCR 檢測 CUEDC2 mRNA 的表達(dá)。western-blot檢測CUEDC2蛋白的表達(dá)。5淀粉酶、TNF-α、IL-6測定:應(yīng)用淀粉酶試劑盒檢測細(xì)胞上清中淀粉酶(amylase,AMY)水平,應(yīng)用 ELISA(enzyme linked immunosorbent assay)方法檢測細(xì)胞培養(yǎng)上清中腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)及白細(xì)胞介素-6(interleukin-6,IL-6)水平。6統(tǒng)計(jì)學(xué)方法:應(yīng)用SPSS 21.0軟件包進(jìn)行統(tǒng)計(jì)學(xué)處理,P0.05認(rèn)為具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1成功建立了急性胰腺炎細(xì)胞模型,雨蛙素組AMY的水平隨時(shí)間延長而增高且均較對照組升高(P0.05)。雨蛙素組中CUEDC2mRNA水平較對照組下降(P0.05)。雨蛙素組中各個(gè)時(shí)間點(diǎn)的CUEDC2mRNA無明顯變化(P0.05);2應(yīng)用CUEDC2質(zhì)粒轉(zhuǎn)染細(xì)胞后,可見綠色熒光顆粒,細(xì)胞生長狀態(tài)良好,表明轉(zhuǎn)染條件穩(wěn)定,細(xì)胞轉(zhuǎn)染成功,轉(zhuǎn)染率約為65%-70%。CUEDC2質(zhì)粒組CUEDC2蛋白水平較對照組升高(P0.05)。陰性質(zhì)粒組與對照組相比未見明顯差異(P0.05);3細(xì)胞培養(yǎng)上清AMY:各個(gè)時(shí)間點(diǎn)比較,在陰性質(zhì)粒對照組和CUEDC2質(zhì)粒對照組中AMY水平未見明顯上升,且兩組之間未見明顯差異(P0.05);在各個(gè)時(shí)間點(diǎn)AMY水平,陰性質(zhì)粒對照組和CUEDC2質(zhì)粒對照之間無顯著差異(P0.05)。在陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組中AMY水平隨時(shí)間逐漸增高(P0.05)。陰性質(zhì)粒雨蛙素組與CUEDC2質(zhì)粒雨蛙素組均較各自對照組升高(P0.05);CUEDC2質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比AMY水平下降(P0.05);4細(xì)胞培養(yǎng)上清TNF-α、IL-6:在陰性質(zhì)粒對照組和CUEDC2質(zhì)粒對照組中各個(gè)時(shí)間點(diǎn)TNF-α、IL-6水平較低,且兩組之間無統(tǒng)計(jì)學(xué)意義(P0.05);在陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組中隨時(shí)間的延長TNF-α、IL-6水平逐漸升高(P0.05)。陰性質(zhì)粒雨蛙素組與CUEDC2質(zhì)粒雨蛙素組在各個(gè)時(shí)間點(diǎn)TNF-α、IL-6水平均較各自對照組顯著上升(P0.05);CUEDC質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比,在各個(gè)時(shí)間點(diǎn)TNF-α、IL-6水平均降低(P0.05)。結(jié)論:1應(yīng)用雨蛙素誘導(dǎo)可成功制備AP細(xì)胞模型。2在AP細(xì)胞模型中CUEDC2 mRNA的水平下調(diào)。3成功構(gòu)建了過表達(dá)CUEDC2蛋白的AP細(xì)胞模型,為研究CEUDC2在急性胰腺炎中的作用奠定了基礎(chǔ)。4過表達(dá)CUEDC2后,雨蛙素誘導(dǎo)的AR42J細(xì)胞產(chǎn)生AMY、TNF-α及IL-6水平降低,說明CUEDC2能抑制炎性介質(zhì)的過度增長,在急性胰腺炎中其保護(hù)作用。
[Abstract]:Acutepancreatitis (AP) is a common clinical acute abdomen, which has the characteristics of acute onset, severe disease and rapid change. In addition to the pancreatic disease, it may also cause injury to the external organs and tissues of the pancreas, and even eventually induce systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), multiple organ dysfunction. Death is caused by multiple-organ dysfunction syndrome (MODS). Although significant progress has been made in the study of acute pancreatitis in the past few decades, its pathogenesis is still more complex. Acute pancreatitis (acute pancreatitis, AP), regardless of the cause of the disease, causes the pancreas to cause the digestion of the pancreatin, as well as the inflammation of the pancreas. The irritant stimulates mononuclear macrophages and pancreatic acinar cells to release inflammatory mediators and cytokines, triggering the inflammatory mediators to form a cascade cascade, which eventually leads to local or systemic inflammatory response.CUEDC2 (CUE domain-containing 2), a multipotent protein containing the CUE domain, which is widely expressed in human tissues and organs. A large number of studies have shown that CUEDC2 can play a role in a variety of intracellular signal transduction pathways involved in many cell activities, such as cell cycle regulation, growth signal transduction, tumor development, and inflammatory reactions. Current studies have found that CUEDC2 is not only closely related to the development of tumors, such as breast cancer, colitis associated tumor, lung Cancer and so on, can also participate in inflammatory reactions, such as colitis, etc. the relationship between CUEDC2 and acute pancreatitis has not been reported. This study intends to preliminarily explore the role of CUEDC2 in acute pancreatitis. Objective: to induce AR42J cells to establish acute pancreatitis cell model by using rain frog hormone and to use plasmid transfection technique. The effects of CUEDC2. on the level of amylase, AMY, tumor necrosis factor- alpha, TNF- alpha, and interleukin -6 (interleukin-6, IL-6) were overexpressed in the cell culture medium, and the effect of CUEDC2 on the TNF- alpha in acinar cells of acute pancreatitis was preliminarily discussed. Methods: 1 using rain frog hormone to induce acute pancreatitis The cell model.2 obtained the AR42J cells that overexpressed CUEDC2: the CUEDC2 plasmid was transferred into AR42J cells by liposome transfection, and the transfection efficiency.3 experimental grouping was examined by fluorescence microscopy: AR42J cells were divided into 4 groups: negative plasmid control group (Neg-Control group), negative plasmid rain frog group (Neg-CAE group), CUEDC2 plasmid control group (CUEDC2-Control) Group of CUEDC2 plasmids (group CUEDC2-CAE). The specific grouping method is as follows: negative plasmid group: transfection of negative plasmid to AR42J cell.Neg-CAE group to give 10-7 mol/L rain frog, Neg-Control group to give equal volume complete medium, and then divide two groups into 3 subgroups: 4h, 8h, 24h.CUEDC2 plasmid group: CUEDC2 plasmid transfection AR42J cell.CUEDC Group 2-CAE was given 10-7mol/L rain frog CUEDC2-Control group CUEDC2-Control group given equal volume complete medium, and then divided into 3 subgroups: 4h, 8 h, 24h.4 CUEDC2 determination: PCR detection CUEDC2 mRNA expression.Western-blot detection of CUEDC2 protein expression of amylase Se, AMY) level, the application of ELISA (enzyme linked immunosorbent assay) method to detect tumor necrosis factor alpha (tumor necrosis factor- a, TNF- alpha) and interleukin -6 (enzyme linked immunosorbent assay). An acute pancreatitis cell model was successfully established. The level of AMY in the rain frog group increased with time and was higher than that in the control group (P0.05). The CUEDC2mRNA level in the rain frog group was lower than that in the control group (P0.05). The CUEDC2mRNA of each time point in the rain frog group was not significantly changed (P0.05); 2 the green fluorescence was visible after the transfection of the CUEDC2 plasmid. The growth condition of the cells was good, which showed that the transfection condition was stable and the transfection was successful. The transfection rate was higher than that of the control group (P0.05), the transfection rate was about 65%-70%.CUEDC2 plasmid group (P0.05). The negative plasmid group had no significant difference compared with the control group (P0.05). The 3 cell culture supernatant AMY: was compared at every time point in the negative plasmid control group and CUEDC2 substance. There was no significant increase in the level of AMY in the control group, and there was no significant difference between the two groups (P0.05). There was no significant difference between the negative plasmid control group and the CUEDC2 plasmid control at the AMY level at each time point (P0.05). The AMY level increased gradually in the negative plasmid rain frog group and the CUEDC2 plasmid wet frog group (P0.05). The group and CUEDC2 plasmid wet frog group were all higher than those of the control group (P0.05), and the AMY level of the CUEDC2 plasmid wet frog group was lower than the negative plasmid (P0.05), and the 4 cell culture supernatant TNF- a, IL-6: in the negative plasmid control group and the CUEDC2 plasmid control group, the TNF- alpha, IL-6 level was lower, and there was no statistical significance between the two groups. (P0.05); the level of TNF- alpha and IL-6 increased gradually in the negative plasmids of the rain frog group and the CUEDC2 plasmid wet frog group (P0.05). The TNF- A and IL-6 levels of the negative plasmids and CUEDC2 plasmids were significantly higher than those of the control group at all time points (P0.05); CUEDC plasmid rain frog group and the negative plasmid, rain frog group. Compared with TNF- alpha at every time point, IL-6 water decreased (P0.05). Conclusion: 1 the AP cell model can be successfully prepared by the induction of the AP cell model.2 in the AP cell model, and the level of CUEDC2 mRNA in the AP cell model is downregulated and.3 successfully constructed the AP cell model for the expression of CUEDC2 protein, which lays a foundation for the study of the role of CEUDC2 in acute pancreatitis. After that, AR42J cells induced by rain frogs produced AMY, TNF- alpha and IL-6 levels decreased, indicating that CUEDC2 can inhibit the excessive growth of inflammatory mediators and its protective effect in acute pancreatitis.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R576

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 申萌;羅正茂;張虹;黃遠(yuǎn)航;李浪;何鳳;童俊容;;CUEDC2對梗阻性腎病小鼠腎組織炎癥反應(yīng)及間質(zhì)纖維化的作用[J];實(shí)用醫(yī)學(xué)雜志;2016年12期

2 ;Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure[J];World Journal of Gastroenterology;2012年36期

3 Jessica K Dyson;Matthew D Rutter;;Colorectal cancer in inflammatory bowel disease:What is the real magnitude of the risk?[J];World Journal of Gastroenterology;2012年29期

4 崔佳梅;張明;張學(xué)敏;顏賢忠;;CUEDC2中CUE結(jié)構(gòu)域的表達(dá)純化及結(jié)構(gòu)初探[J];生物技術(shù)通訊;2011年04期

5 劉志鵬;鄒利權(quán);游斌;;血清TNF-α、sTNF-1R及IL-10在急性胰腺炎中的變化及意義[J];重慶醫(yī)學(xué);2011年05期

6 Marja-Leena Kylnp;Heikki Repo;Pauli Antero Puolakkainen;;Inflammation and immunosuppression in severe acute pancreatitis[J];World Journal of Gastroenterology;2010年23期

7 王峰;李建生;王守富;李亞;劉四化;張會(huì)儉;秦金麗;;阻斷JAK/STAT信號轉(zhuǎn)導(dǎo)通路對克雷伯桿菌肺炎老齡大鼠生化和病理的影響[J];中國中醫(yī)藥現(xiàn)代遠(yuǎn)程教育;2008年03期

8 張喜平;吳承鈞;李志軍;;重癥急性胰腺炎并發(fā)肺損傷的研究進(jìn)展[J];世界華人消化雜志;2008年03期

9 鄭仲謹(jǐn);核轉(zhuǎn)錄因子κB在全身炎癥反應(yīng)綜合征中的作用[J];免疫學(xué)雜志;2001年04期

相關(guān)碩士學(xué)位論文 前1條

1 周維;CUEDC2/p-IKKβ/NF-κB在膽管癌中表達(dá)與臨床意義研究[D];山西醫(yī)科大學(xué);2015年

，

本文編號：1886951