本文關(guān)鍵詞：Pim-3，occludin及ICAM-1在受損腸黏膜中的表達(dá)規(guī)律和相關(guān)性的實(shí)驗(yàn)研究　出處：《南昌大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Pim-3基因 燒傷 腸機(jī)械屏障 脂多糖

【摘要】： 一、研究背景 腸道是體內(nèi)最大的貯菌庫和內(nèi)毒素庫,在正常情況下,腸屏障能阻止腸道內(nèi)細(xì)菌及其分解產(chǎn)物經(jīng)腸壁擴(kuò)散至機(jī)體內(nèi),但是,在嚴(yán)重感染、創(chuàng)傷、休克等應(yīng)激狀況下,腸道屏障功能受損,腸道內(nèi)微生物和內(nèi)毒素可通過受損的腸粘膜屏障侵入到體循環(huán),形成腸源性感染,導(dǎo)致多器官功能障礙。因此,腸屏障功能已成為判斷危重患者預(yù)后的重要指標(biāo)之一。也正因如此,如何有效保護(hù)腸黏膜屏障功能成為當(dāng)前消化領(lǐng)域的研究熱點(diǎn)之一。以前這方面的研究思路主要集中在腸內(nèi)營養(yǎng)及恢復(fù)腸內(nèi)正常微生態(tài)環(huán)境上,而利用原癌基因治療腸黏膜損傷卻少有涉及,對(duì)燒傷及內(nèi)毒素作用后腸道生物屏障的變化及其與原癌基因的相關(guān)性亦較少見報(bào)道。以往的研究證明,很多原癌基因在組織受損后都具有增量調(diào)節(jié)的作用。例如,c-fos、c-jun、egr-1、sp-1、表皮生長因子、轉(zhuǎn)化生長因子、成纖維細(xì)胞生長因子、血管內(nèi)皮生長因子,所有這些基因產(chǎn)物都扮演著一個(gè)重要的修補(bǔ)角色,協(xié)助修復(fù)受損組織。原癌基因Pim-3編碼`一種絲/蘇氨酸蛋白激酶Pim-3,它廣泛表達(dá)于人類各種組織細(xì)胞中,可促進(jìn)MAPK磷酸化,參與多個(gè)細(xì)胞信號(hào)通路的調(diào)控,,它具有促進(jìn)細(xì)胞生長和抑制凋亡的功能,在損傷組織的修復(fù)、治療中發(fā)揮積極作用。為此我們?cè)O(shè)想,Pim-3可能有修復(fù)腸黏膜損傷,保護(hù)腸道機(jī)械屏障功能的作用。而腸上皮細(xì)胞間的緊密連接是由緊密連接分子來完成,occludin就是其中的一個(gè)重要成員,并在腸道機(jī)械屏障中發(fā)揮重要作用。Fujibe等發(fā)現(xiàn)MAPK參與緊密連接分子occludin、Claudins等的表達(dá)調(diào)控。另外,ICAM-1作為黏附分子,對(duì)淋巴細(xì)胞和中性粒細(xì)胞進(jìn)入腸道有趨化、介導(dǎo)作用,一方面參與腸道免疫屏障功能,另一方面也促進(jìn)了腸道的炎癥反應(yīng)。這已被大量的研究所證實(shí)。為此,本研究以30%TBSA燒傷大鼠及內(nèi)毒素?fù)p傷大鼠為模型,對(duì)燒傷或內(nèi)毒素引起腸黏膜損傷后相關(guān)基因(Pim-3、occludin、ICAM-1)的表達(dá)進(jìn)行系統(tǒng)觀察,研究它們的表達(dá)規(guī)律并分析彼此間的相關(guān)性。為后續(xù)實(shí)驗(yàn)提供實(shí)驗(yàn)數(shù)據(jù)和理論支持。 二、總體思路 首先,選用健康Wistar大鼠120只,雌雄各半,體重220±20g,按隨機(jī)表隨機(jī)分為四組:A組燒傷組,B組LPS組, C組.燒傷對(duì)照組,D組.LPS對(duì)照組。每組30只,各組均于處理后3h、6h、12h、24h、48h開腹取小腸組織,每組各時(shí)間點(diǎn)均為6只動(dòng)物。建立30%TBSAⅢ燒傷大鼠模型及內(nèi)毒素大鼠模型,取燒傷皮膚做病理檢查以確定燒傷程度,在各時(shí)間點(diǎn)取小腸組織,一部分做病理檢查以確定腸黏膜損傷程度,另一部分用來檢測(cè)Pim-3、閉鎖蛋白(Occludin)以及細(xì)胞間黏附分子(ICAM-1)的mRNA和蛋白水平,了解燒傷或內(nèi)毒素處理后48小時(shí)內(nèi)Pim-3、occludin及ICAM-1的表達(dá)規(guī)律,并分析它們之間的相關(guān)性。 三、實(shí)驗(yàn)?zāi)康�、方法、結(jié)果及結(jié)論 1.目的通過建立成年大鼠30%TBSAⅢ燒傷模型和內(nèi)毒素大鼠模型,利用RT-PCR及western blotting等分子生物學(xué)方法檢測(cè)Pim-3 occludin及ICAM-1在損傷腸黏膜中的表達(dá)變化。研究它們的表達(dá)規(guī)律并分析彼此間的相關(guān)性。為后續(xù)實(shí)驗(yàn)提供實(shí)驗(yàn)數(shù)據(jù)和理論支持。 2.方法將實(shí)驗(yàn)大鼠隨機(jī)分為四組:A組.燒傷組(n=30);B組.內(nèi)毒素(LPS)組(n=30);C組.燒傷對(duì)照組,D組.LPS對(duì)照組。各組依次在處理后3h、6h、12h、24h、48h取小腸組織,一部分進(jìn)行常規(guī)HE染色及病理切片檢查,另一部分進(jìn)行RT-PCR及Western blotting,分別檢測(cè)內(nèi)源性Pim-3、Occludin、ICAM-1在腸道的表達(dá)變化。 3.結(jié)果⑴、燒傷皮膚的病理改變:大鼠燒傷區(qū)皮膚表皮缺如,真皮及皮下組織出現(xiàn)凝固性壞死,部分皮下肌肉組織出現(xiàn)肌溶解。⑵、小腸粘膜的病理改變:經(jīng)HE染色,可見燒傷后3 h腸絨毛出現(xiàn)水腫,內(nèi)皮脫落,粘膜固有層充血,炎性細(xì)胞浸潤,以后幾個(gè)時(shí)間點(diǎn)腸黏膜的病理改變與上面的相仿,沒有大的很明顯的變化,傷后48 h腸粘膜病變還未恢復(fù)正常。與燒傷組相比,內(nèi)毒素組小腸粘膜病理改變更為明顯,正常對(duì)照組無明顯變化;⑶、Pim-3的檢測(cè)結(jié)果:正常大鼠小腸組織Pim-3 mRNA和蛋白水平低表達(dá),而燒傷組和內(nèi)毒素組在3 h表達(dá)開始增多,6 h到達(dá)頂點(diǎn),12 h開始下降,24 h、48 h低表達(dá)。燒傷組、內(nèi)毒素組內(nèi)源性Pim-3基因表達(dá)與各自對(duì)照組之間的差異有統(tǒng)計(jì)學(xué)意義(p㩳0.01),燒傷組3 h、6 h、12 h、24 h、48 h分別為對(duì)照組的12.25±1.24,21.13±1.78,14.21±1.12,2.12±0.11,1.08±0.07倍;內(nèi)毒素組分別為對(duì)照組的15.08±1.07,25.24±2.11,16.32±1.23,2.15±0.13, 1.10±0.08倍;燒傷組、內(nèi)毒素組之間的差異無統(tǒng)計(jì)學(xué)意義(p0.05)。⑷occludin的檢測(cè)結(jié)果:在燒傷組,western blotting結(jié)果證實(shí)occludin蛋白水平在3小時(shí)和6小時(shí)升高。RT-PCR方法顯示,燒傷后3 h、6 h、12 h、24 h、48 h閉鎖蛋白mRNA量升高,分別為對(duì)照組的3.25±0.26、6.65±0.44.、3.10±0.25, 1.55±0.08, 1.01±0.08倍;內(nèi)毒素組3 h、6 h、12 h、24 h、48 h分別為對(duì)照組的3.15±0.25, 6.25±0.56, 2.52±0.18, 1.42±0.10, 1.05±0.08倍(P㩳0.05)。內(nèi)毒素組與燒傷組比較經(jīng)統(tǒng)計(jì)學(xué)檢驗(yàn)無顯著性差異(P0.05)。⑸、ICAM-1的檢測(cè)結(jié)果:燒傷及內(nèi)毒素處理后ICAM-1 mRNA量增加,以12h升高明顯( P0.01 )。燒傷組3h、6h、12h、24h、48h為對(duì)照組的12.12±1.32, 18.16±1.68,19.31±1.74,15.53±1.46,12.12±1.12倍(P㩳0.05);內(nèi)毒素組3 h、6 h、12 h、24 h , 48h分別為對(duì)照組的12.81±1.12,19.68±1.67 ,17.43±1.55, 16.24±1.41, 18.12±1.61倍(P㩳0.05)。燒傷組、內(nèi)毒素組ICAM-1 mRNA的表達(dá)與對(duì)照組之間的差異有統(tǒng)計(jì)學(xué)意義(p㩳0.05);內(nèi)毒素組與燒傷組比較經(jīng)統(tǒng)計(jì)學(xué)檢驗(yàn)無顯著性差異(P0.05)。 4.結(jié)論在燒傷及內(nèi)毒素作用下,Pim-3和occludin在腸粘膜損傷的早期(6h內(nèi))同時(shí)出現(xiàn)高表達(dá),隨后又都逐漸降低,變化曲線大致相同,這表明它們之間以及它們與腸粘膜損傷之間存在相關(guān)性。而ICAM-1一直出現(xiàn)高表達(dá),表明與處理因素存在相關(guān)性,與Pim-3和occludin則無明顯關(guān)聯(lián)。
[Abstract]:First, research background
The intestinal tract is the body's largest reservoir of bacteria and endotoxin in the library library, under normal circumstances, the intestinal barrier can prevent intestinal bacteria and its decomposition products through the intestinal mucosa into the body, but in severe infection, trauma, shock stress, intestinal barrier dysfunction, intestinal microbial and endotoxin can invade the body cycle through the intestinal mucosal barrier is damaged, the formation of enterogenic infection, leading to multiple organ dysfunction. Therefore, intestinal barrier function has become an important indicator to determine the prognosis of critically ill patients. Because of this, how to protect the intestinal mucosal barrier function effectively has become one of the research hotspots in the field of digestion. Previous research ideas in this field mainly focus enteral nutrition in recovery and normal intestinal micro ecological environment, and the use of the original cancer gene therapy of intestinal mucosa injury was less involved, the changes of intestinal barrier function after burn and endotoxin And its correlation with the original cancer gene is rarely reported. Previous studies showed that many oncogenes have incremental regulation role in tissue damage. For example, c-fos, c-jun, Egr-1, SP-1, epidermal growth factor, transforming growth factor, fibroblast growth factor, vascular endothelial growth factor, all of these genes the product plays an important role to help repair, repair damaged tissue. Pim-3 gene, encoding a serine / threonine protein kinase Pim-3, which is widely expressed in various human tissues and cells, promoting MAPK phosphorylation, regulation, participate in multiple cell signaling pathways, it can promote cell growth and inhibition apoptosis, repair of tissue damage, play a positive role in the treatment. We hypothesized that Pim-3 may have intestinal mucosa damage repair, protect the intestinal mechanical barrier function and intestinal epithelial cells. The tension between the Close connection is made by occludin to complete, one is one of the important members of the occludin, and in the intestinal mechanical barrier plays an important role in the.Fujibe and found that MAPK involved in occludin expression and regulation of Claudins and occludin. In addition, ICAM-1 as adhesion molecules on lymphocytes and neutrophils into the gut mediated chemotaxis the leading role of a part in the intestinal immune barrier function, but also can promote the intestinal inflammation. It has been proved by a lot of research. Therefore, this research is based on the 30%TBSA of burn rats and endotoxin injury in rats caused by intestinal mucosal injury after genes related to burn or endotoxin (Pim-3, occludin, ICAM-1) were observed the expression, expression of them and to analyze the correlation between each other. To provide experimental data and theoretical support for subsequent experiments.
Two, the general idea
First of all, selected 120 healthy Wistar rats, male and female, weight 220 + 20g, were randomly divided into four groups: A group B group burn group, LPS group, C group. Burn control group, D group and.LPS control group. Each group had 30 rats, each group after treatment 3h, 6h, 12h 24h, 48h, open small intestine, each time point was 6. The animal model of 30%TBSA was established with rat burn model and endotoxin in rats, the burn skin pathological examination to determine the degree of burn, small intestine at each time point, a part of pathological examination to determine the degree of injury of intestinal mucosa, the other one part is used to detect Pim-3 protein (Occludin) and atresia of intercellular adhesion molecule (ICAM-1) mRNA and protein levels, about 48 hours after the treatment of burns or endotoxin Pim-3, expression of occludin and ICAM-1, and analyze the correlation between them.
Three, experimental purposes, methods, results and conclusions
The 1. is to establish the adult rat 30%TBSA III burn model and endotoxin rat model, expression of Western using RT-PCR and blotting molecular biology method for detection of Pim-3 occludin and ICAM-1 in intestinal mucosa injury. Expression of them and analyze the relationship between each other. To provide experimental data and theoretical support for subsequent experiments.
Methods 2. rats were randomly divided into four groups: A group (n=30). The burn group; B group. Endotoxin (LPS) group (n=30); C group. Burn control group, D group and.LPS control group. Each group in turn after treatment 3h, 6h, 12h, 24h, 48h of small intestine. A part of the physical section examination of routine HE staining and the disease, another part of the RT-PCR and Western blotting, were used to detect endogenous Pim-3, Occludin, ICAM-1 expression in the gut.
3. results, pathological changes of the rat burn skin: skin burn area of dermis and subcutaneous tissue necrosis, part of subcutaneous tissue appeared muscle dissolved. Ti, pathological changes of the intestinal mucosa: by HE staining, visible burn after 3 h of intestinal villi edema, endothelial denudation, lamina propria congestion and the infiltration of inflammatory cells and the pathological changes of intestinal mucosa and above after several time points were not big, obvious change, 48 h after the injury of intestinal mucosal lesions has not yet returned to normal. Compared with the burn group, endotoxin group of Small Intestinal Mucosal pathological change is more obvious, the normal control group had no obvious change; "the detection results of Pim-3, low expression in normal rat intestinal tissue Pim-3 mRNA and protein levels, and burn group and endotoxin group in 3 h expression began to increase, reached a peak of 6 h, 12 h, 24 h, 48 began to decline, h low expression. Burn group, endogenous endotoxin group 鎬im-3鍩哄洜琛ㄨ揪涓庡悇鑷鐓х粍涔嬮棿鐨勫樊寮傛湁緇熻瀛︽剰涔，

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