【作者】 张磊；

【导师】 刘来亭； 黄进；

【作者基本信息】 河南工业大学 ， 动物营养与饲料科学， 2016， 硕士

【摘要】 转化生长因子β(TGF-β)是一种具有能够提高黏膜修复能力的上皮生长因子,在肠道损伤修复中发挥着重要的作用,在黏膜损伤后能促进上皮黏膜修复。本试验旨在观察TGF-β/Smads信号通路关键因子在雏鸡肠黏膜损伤中的表达,探讨其在肠黏膜损伤中所起的作用。本试验以罗曼公雏为研究对象,通过腹腔注射大肠杆菌和灌喂柔嫩艾美耳球虫卵囊,分别诱导雏鸡的急性和慢性肠道损伤。一方面,测定免疫器官指数和血清炎症因子含量,对受损肠道中部分炎症因子和紧密连接蛋白的基因表达进行检测;另一方面,对肠道损伤模型中TGF-β/Smads信号通路关键因子的基因表达进行检测,Western Blot检测部分因子蛋白的表达。初步揭示TGF-β/Smads信号通路在肠道损伤时各组织中的表达与作用。为下一步深入研究TGF-β/Smads信号通路在肠黏膜损伤中的作用机制奠定基础。实验一:TGF-β/Smads信号通路在大肠杆菌诱导雏鸡急性肠黏膜损伤中的作用选择初始体重无差异的6日龄雏鸡72只,随机分成对照组和E.coli组两个处理,每处理6个重复,每重复6只鸡。E.coli组每只腹腔注射1mL浓度为6×109cfu/mL的大肠杆菌菌悬液;对照组每只注射等体积的无菌LB培养液。注射6h后每重复各取出1只鸡,处死,解剖采集空肠,提取总RNA,反转录后,检测炎症因子IL-1β、IL-6、TNF-α,紧密连接蛋白ZO-1、2,以及TGF-β/Smads信号通路中关键因子TGF-β2、3、4,TβRⅠ、Ⅱ和Smad2、3、7的基因表达。试验结果表明,受损肠道的IL-6基因在炎症初期的表达被抑制,IL-1β基因表达显著上升(P<0.05),肠道的ZO-1、2基因表达未显著改变(P>0.05),说明通透性未发生改变;TGF-β4的基因表达显著上升,伴随着TGF-β2、3的显著降低(P<0.05),TβRⅠ、Ⅱ基因表达量变化不显著(P>0.05),Smad 3基因表达量变化不显著(P>0.05),Smad2、7基因表达量显著下降(P<0.05)。实验二:TGF-β/Smads信号通路在球虫诱导仔鸡慢性肠道损伤中的作用(一)炎症因子和紧密连接蛋白基因的表达及血清中炎症因子的含量测定16日龄雏鸡120只,随机分成对照组和球虫组两个处理,每处理6个重复,每重复10只鸡。球虫组每只灌胃105个卵囊,对照组灌喂等体积生理盐水。分别在感染后6h,72h,第5、6、8天每重复分别取出1只鸡处死,采集脾脏、法氏囊,记录免疫器官指数,采集盲肠提取总RNA,反转录后检测炎症因子IL-1β、IL-6、TNF-α、TLR4,和紧密连接蛋白ZO-1、2的基因表达。试验结果显示,球虫组雏鸡脾脏相对重量在感染后第5、6天增重明显(P<0.05)。感染6h后,盲肠TLR4、IL-6基因表达显著下降(P<0.05);感染第5天,TNF-α、IL-1β显著上升;感染第8天,TNF-α、IL-6显著上升(P<0.05)。盲肠ZO-1基因表达在感染后6h,第5、6、8天一直明显下降,ZO-2基因表达在感染后6h显著下降,第5、6、8天显著上升(P<0.05)。这些结果提示我们在这些时间点盲肠通透性增加。我们采集感染后第6天和第8天雏鸡的血清,使用Elisa试剂盒对IFN-γ、IL-1β、IL-6、IL-12 p40的含量进行检测,发现对照组和球虫组间差异不显著(P>0.05)。(二)TGF-β/Smads信号通路中各关键因子基因的表达及部分因子蛋白含量的测定分别在感染后6h,72h,第5、6、8天每重复分别取出1只鸡处死,采集脾脏、肝脏、盲肠,提取各组织总RNA,反转录后检测TGF-β2、3、4,TβRⅠ、Ⅱ,Smad2、3、7的基因表达,Western Blot检测盲肠TGF-β2、3,Smad2的相对蛋白含量变化。研究结果表明,盲肠TGF-β2基因表达在感染后6h、第5、6、8天均显著下降(P<0.05),TGF-β3在感染后6h和第6天显著下降(P<0.05),TGF-β4则是在72h和第8天显著下降(P<0.05),TβRⅠ、Ⅱ基因表达在6h、第5、6天也显著下降(P<0.05),Smad2在6h、72h显著下降(P<0.05),Smad3在6h显著下降,第6天则显著上升(P<0.05);脾脏TGF-β2在感染后6h和第6天显著下降(P<0.05),TGF-β3则在第5、6天显著下降(P<0.05),在第8天显著上升(P<0.05),TGF-β4基因表达在感染后72h显著下降后,在第5天显著上升(P<0.05),Smad2、3基因表达在感染后6h显著下降,第5天又显著上升(P<0.05),在感染后第8天恢复正常水平;肝脏的TGF-β2、3,TβRⅠ、Ⅱ,Smad2、7基因表达在感染后第8天均显著上升(P<0.05),在之前的时间点上,TGF-β2的基因表达在感染后72h显著上升,第6天显著下降(P<0.05);TβRⅠ在感染后72h和第6天显著上升(P<0.05);Smad3在感染后第6天显著上升(P<0.05)。感染后6h、72h、第6天与第8天的TGF-β2、3,Smad2的相对蛋白含量没有出现显著变化(P>0.05)。更多还原

【Abstract】 Transforming growth factor-beta(TGF-β) is an epidermal growth factor which can improve the mucosal repair capacity, and plays an important role in the repair of intestinal injury. This experiment was designed to investigate the expression of the key factos of TGF-β/Smads signaling pathway in the chicken’s injury intestinal mucosal, and the role they played in intestinal mucosal injury.In this experiment, egg-type cockerels(Roman) were used to study. The acute and chronic intestinal injury were induced by injecting chicks E.coli and feeding chicks Eimeria tenella oocysts. First, the immune organ index and inflammatory cytokines in serum were measured, the expression of inflammatory cytokines and tight junction proteins in the injury gut were also detected; we detected the expression of the key factors of TGF-β/Smads signalinging pathway in the injury gut, spleen and liver, and part of factors’ proteins were detected by Western Blot. We would like to reveal the expression and funcation of TGF-β/Smads signaling pathway in different organs when chicken gets an intestinal mucosal injury, and prepare to make a futther and deeper study of TGF-β/Smads signaling pathway in intestinal mucosal injury.Experiment 1: The role of TGF-β/Smads signaling pathway in chicken acute mucosal injury induced by E.coli72 6-day-old chicks were randomly divided into control group and E.coli group,each group had 6 replicates with 6 chicks. Each chick of E.coli group was injected1 m L E.coli bacterial suspension with a concentration of 6×109 cfu /m L; the control group were injected with an equal volume sterile LB broth.6 hours after injection, 1chick from each replicates were chosen to be killed, the jejunum was collected, then the total RNA was extracted, after reverse transcription, we detected the gene expression of the inflammatory cytokines IL-1β, IL-6, TNF-α,tight junction proteins ZO-1,2,the key factors of TGF-β/Smads signalinging pathway TGF-β2,3,4,TβRⅠ,Ⅱ, Smad2,3,7. The experiment result showed that IL-6 gene expression was suppressed in early inflammation, IL-1β gene expression significantly increase(P<0.05). ZO-1, 2 gene expression didn’t change significantly(P>0.05), which meand the permeability was not changed. TGF-β4’s gene expression increased significantly(P<0.05), accompanied by a significant decrease(P<0.05) of TGF-β2,3. TβR Ⅰ, Ⅱand Smad 3 had no significant change, but the gene expression of Smad2,3 decreased significantly(P<0.05).Experiment 2: The role of TGF-β/Smads signaling pathway in chicken chronic mucosal injury induced by E.tenellaPart 1:The expression of inflammatory cytokines and tight junction proteins and the content of inflammatory cytokines in serum120 16-day-old chicks were randomly divided into control group and E.tenella group, each group had 6 replicates with 10 chicks. Each chick of E.tenella group was fed 105 oocysts, the control group was fed with normal saline. In 6h, 72 h, 5th day, 6th day and 8th day after infection, 1 chick from each replicates were chosen to be killed,spleen、 bursa were collected and record immune organs index. Cecal was collected,and total RNA was extracted, after reverse transcriptase, we detected the expression of the inflammatory cytokines IL-1β, IL-6, TNF-α, TLR4 and tight junction proteins ZO-1,2.The result showed that the spleen’s weight was increased significantly(P<0.05) on 5th and 6th day after infection. TLR4 and IL-6 gene expression decreased significantly(P<0.05) in 6h after infection; TNF-α and IL-1βgene expression increased significantly(P<0.05) on 5th day after infection; TNF-α and IL-6 gene expression increased significantly(P<0.05) on 8th day after infection. ZO-1 gene expression decreased significantly(P<0.05) in 6h and 5th 、 6th 、 8thday, ZO-2 geneexpression decreased significantly(P<0.05) in 6h, but increased significantly(P<0.05)on 5th,6th,8th day after infection. We collected chichen’s serum on 6th and 8th day after injection, and used Elisa kit to check IFN-γ,IL-1β,IL-6,IL-12 p40’s contents, but we found no significant difference between control group and E. tenella group(P>0.05).Part 2:The expression of the key factors of TGF-β/Smads signaling pathway in the injury gut and other organs, and the content of some factors’ proteins:In 6h, 72 h, 5th day, 6th day and 8th day after infection, 12 chicks of each group were killed. Spleen, liver, cecum were collected, and total RNA was extracted, after reverse transcriptase, we detected the expression of TGF-β2,3,4, TβR Ⅰ, Ⅱ,Smad2,3,7 in these organs. And TGF-β2, 3, Smad2’s proteins’ content were detected by Wstern Blot. The result showed that TGF-β2 gene expression in cecum decreased significantly(P<0.05) in 6h,5th,6th,8th day after infection, TGF-β3 decreased significantly(P<0.05) in 6h,6th day, TGF-β4 decreased significantly(P<0.05) in 72 h,8th day, so did TβRⅠ,Ⅱ in 6h,5th,6th day(P<0.05). TGF-β2 gene expression in spleen decreased significantly(P<0.05) in 6h, 6th day, TGF-β3 decreased significantly(P<0.05) in 5th, 6th day, and increased significantly(P<0.05) in 8th day, TGF- β4 gene expression decreased significantly(P<0.05) in 72 h, and increased significantly(P<0.05) on 5th day, Smad2, 3 gene expression increased at first and decreased then,returned to normal level finally. TGF- β2,3, TβRⅠ,Ⅱ, Smad2,7 gene expression in liver increased significantly(P<0.05) on 8th day, before this time, the gene expression of TGF-β2 had a significantly increased in 72 h after infection, and a significantly decreased on 6th day(P<0.05). The gene expression of TβR Ⅰ had a significantly increased in 72 h and 6th day( P<0.05). The gene expression of Smad3 had a significantly increased on 6th day( P<0.05). The content of TGF-β2, 3, Smad2’s proteins had no significantly change in 6h,72 h,6th and 8th day after infection(P>0.05).更多还原