Pim-3，occludin及ICAM-1在受损肠黏膜中的表达规律和相关性的实验研究

发布时间：2018-01-02 04:13

  本文关键词：Pim-3，occludin及ICAM-1在受损肠黏膜中的表达规律和相关性的实验研究　出处：《南昌大学》2008年硕士论文　论文类型：学位论文

  更多相关文章： Pim-3基因 烧伤 肠机械屏障 脂多糖

【摘要】： 一、研究背景 肠道是体内最大的贮菌库和内毒素库,在正常情况下,肠屏障能阻止肠道内细菌及其分解产物经肠壁扩散至机体内,但是,在严重感染、创伤、休克等应激状况下,肠道屏障功能受损,肠道内微生物和内毒素可通过受损的肠粘膜屏障侵入到体循环,形成肠源性感染,导致多器官功能障碍。因此,肠屏障功能已成为判断危重患者预后的重要指标之一。也正因如此,如何有效保护肠黏膜屏障功能成为当前消化领域的研究热点之一。以前这方面的研究思路主要集中在肠内营养及恢复肠内正常微生态环境上,而利用原癌基因治疗肠黏膜损伤却少有涉及,对烧伤及内毒素作用后肠道生物屏障的变化及其与原癌基因的相关性亦较少见报道。以往的研究证明,很多原癌基因在组织受损后都具有增量调节的作用。例如,c-fos、c-jun、egr-1、sp-1、表皮生长因子、转化生长因子、成纤维细胞生长因子、血管内皮生长因子,所有这些基因产物都扮演着一个重要的修补角色,协助修复受损组织。原癌基因Pim-3编码`一种丝/苏氨酸蛋白激酶Pim-3,它广泛表达于人类各种组织细胞中,可促进MAPK磷酸化,参与多个细胞信号通路的调控,,它具有促进细胞生长和抑制凋亡的功能,在损伤组织的修复、治疗中发挥积极作用。为此我们设想,Pim-3可能有修复肠黏膜损伤,保护肠道机械屏障功能的作用。而肠上皮细胞间的紧密连接是由紧密连接分子来完成,occludin就是其中的一个重要成员,并在肠道机械屏障中发挥重要作用。Fujibe等发现MAPK参与紧密连接分子occludin、Claudins等的表达调控。另外,ICAM-1作为黏附分子,对淋巴细胞和中性粒细胞进入肠道有趋化、介导作用,一方面参与肠道免疫屏障功能,另一方面也促进了肠道的炎症反应。这已被大量的研究所证实。为此,本研究以30%TBSA烧伤大鼠及内毒素损伤大鼠为模型,对烧伤或内毒素引起肠黏膜损伤后相关基因(Pim-3、occludin、ICAM-1)的表达进行系统观察,研究它们的表达规律并分析彼此间的相关性。为后续实验提供实验数据和理论支持。 二、总体思路 首先,选用健康Wistar大鼠120只,雌雄各半,体重220±20g,按随机表随机分为四组:A组烧伤组,B组LPS组, C组.烧伤对照组,D组.LPS对照组。每组30只,各组均于处理后3h、6h、12h、24h、48h开腹取小肠组织,每组各时间点均为6只动物。建立30%TBSAⅢ烧伤大鼠模型及内毒素大鼠模型,取烧伤皮肤做病理检查以确定烧伤程度,在各时间点取小肠组织,一部分做病理检查以确定肠黏膜损伤程度,另一部分用来检测Pim-3、闭锁蛋白(Occludin)以及细胞间黏附分子(ICAM-1)的mRNA和蛋白水平,了解烧伤或内毒素处理后48小时内Pim-3、occludin及ICAM-1的表达规律,并分析它们之间的相关性。 三、实验目的、方法、结果及结论 1.目的通过建立成年大鼠30%TBSAⅢ烧伤模型和内毒素大鼠模型,利用RT-PCR及western blotting等分子生物学方法检测Pim-3 occludin及ICAM-1在损伤肠黏膜中的表达变化。研究它们的表达规律并分析彼此间的相关性。为后续实验提供实验数据和理论支持。 2.方法将实验大鼠随机分为四组:A组.烧伤组(n=30);B组.内毒素(LPS)组(n=30);C组.烧伤对照组,D组.LPS对照组。各组依次在处理后3h、6h、12h、24h、48h取小肠组织,一部分进行常规HE染色及病理切片检查,另一部分进行RT-PCR及Western blotting,分别检测内源性Pim-3、Occludin、ICAM-1在肠道的表达变化。 3.结果⑴、烧伤皮肤的病理改变:大鼠烧伤区皮肤表皮缺如,真皮及皮下组织出现凝固性坏死,部分皮下肌肉组织出现肌溶解。⑵、小肠粘膜的病理改变:经HE染色,可见烧伤后3 h肠绒毛出现水肿,内皮脱落,粘膜固有层充血,炎性细胞浸润,以后几个时间点肠黏膜的病理改变与上面的相仿,没有大的很明显的变化,伤后48 h肠粘膜病变还未恢复正常。与烧伤组相比,内毒素组小肠粘膜病理改变更为明显,正常对照组无明显变化;⑶、Pim-3的检测结果:正常大鼠小肠组织Pim-3 mRNA和蛋白水平低表达,而烧伤组和内毒素组在3 h表达开始增多,6 h到达顶点,12 h开始下降,24 h、48 h低表达。烧伤组、内毒素组内源性Pim-3基因表达与各自对照组之间的差异有统计学意义(p�d0.01),烧伤组3 h、6 h、12 h、24 h、48 h分别为对照组的12.25±1.24,21.13±1.78,14.21±1.12,2.12±0.11,1.08±0.07倍;内毒素组分别为对照组的15.08±1.07,25.24±2.11,16.32±1.23,2.15±0.13, 1.10±0.08倍;烧伤组、内毒素组之间的差异无统计学意义(p0.05)。⑷occludin的检测结果:在烧伤组,western blotting结果证实occludin蛋白水平在3小时和6小时升高。RT-PCR方法显示,烧伤后3 h、6 h、12 h、24 h、48 h闭锁蛋白mRNA量升高,分别为对照组的3.25±0.26、6.65±0.44.、3.10±0.25, 1.55±0.08, 1.01±0.08倍;内毒素组3 h、6 h、12 h、24 h、48 h分别为对照组的3.15±0.25, 6.25±0.56, 2.52±0.18, 1.42±0.10, 1.05±0.08倍(P�d0.05)。内毒素组与烧伤组比较经统计学检验无显著性差异(P0.05)。⑸、ICAM-1的检测结果:烧伤及内毒素处理后ICAM-1 mRNA量增加,以12h升高明显( P0.01 )。烧伤组3h、6h、12h、24h、48h为对照组的12.12±1.32, 18.16±1.68,19.31±1.74,15.53±1.46,12.12±1.12倍(P�d0.05);内毒素组3 h、6 h、12 h、24 h , 48h分别为对照组的12.81±1.12,19.68±1.67 ,17.43±1.55, 16.24±1.41, 18.12±1.61倍(P�d0.05)。烧伤组、内毒素组ICAM-1 mRNA的表达与对照组之间的差异有统计学意义(p�d0.05);内毒素组与烧伤组比较经统计学检验无显著性差异(P0.05)。 4.结论在烧伤及内毒素作用下,Pim-3和occludin在肠粘膜损伤的早期(6h内)同时出现高表达,随后又都逐渐降低,变化曲线大致相同,这表明它们之间以及它们与肠粘膜损伤之间存在相关性。而ICAM-1一直出现高表达,表明与处理因素存在相关性,与Pim-3和occludin则无明显关联。
[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�Pim-3鍩哄洜琛ㄨ揪涓庡悇鑷�瀵圭収缁勪箣闂寸殑宸�寮傛湁缁熻�″�︽剰涔，

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