本文選題：隱窩細(xì)胞 + DC��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：家兔腸道易受感染,進(jìn)而導(dǎo)致嚴(yán)重的炎癥和隨后高的死亡率。腸道中的細(xì)菌包括各種共生菌代謝產(chǎn)物和菌體成分可以誘導(dǎo)腸道炎癥反應(yīng)。研究證實(shí)革蘭氏陰性菌菌體比陽(yáng)性菌菌體更容易引起炎癥。與豬和雞等單胃動(dòng)物不同,采食后的家兔各腸段的主要菌門是革蘭氏陰性菌擬桿菌,且革蘭氏陽(yáng)性菌乳酸菌在總菌中所占比例很低。本論文比較健康成年豬、雞和家兔回腸總菌對(duì)培養(yǎng)的家兔隱窩細(xì)胞及DC炎癥反應(yīng)的誘導(dǎo)作用,并研究本實(shí)驗(yàn)室已經(jīng)篩選的幾株兔源乳酸菌對(duì)腸道上皮中隱窩細(xì)胞和樹突狀細(xì)胞的炎性調(diào)節(jié)作用,以探討家兔特有的腸道菌群是否會(huì)導(dǎo)致其腸道更易發(fā)生炎癥,以及乳酸菌是否可緩解炎性反應(yīng),且不同乳酸菌的緩解作用是否存在差異,兩種不同細(xì)胞類型對(duì)抗原的炎性反應(yīng)的差異,為家兔篩選益生菌,提高仔兔免疫力和減少仔兔腸炎發(fā)生提供理論依據(jù)。主要的研究?jī)?nèi)容及結(jié)果如下:1.試驗(yàn)一從未飼喂抗菌素的健康6月齡關(guān)中黑豬、42日齡科寶肉雞和60日齡成年獺兔回腸中收集內(nèi)容物,每種動(dòng)物3個(gè)個(gè)體的回腸內(nèi)容物等量混樣提取菌群基因組DNA,Illumina HiSeq測(cè)序?qū)Ρ确治鲐i、雞和家兔回腸總菌組成。結(jié)果顯示,家兔回腸菌群第一優(yōu)勢(shì)菌門是擬桿菌門,占總菌群的百分比為89.32%;豬和雞回腸菌群最優(yōu)菌門是厚壁菌門,分別占80.79%和88.50%。三種動(dòng)物回腸內(nèi)容物中乳桿菌屬豐度分別為19.19%、81.19%和0.72%。Beta多樣性指數(shù)顯示豬和雞回腸菌群組成接近,家兔與兩者差異較大。2.試驗(yàn)二(1)對(duì)試驗(yàn)一中豬、雞及家兔回腸總菌滅活后作用于培養(yǎng)的家兔隱窩細(xì)胞,空白對(duì)照組添加PBS,熱滅活總菌處理隱窩6 h后收集細(xì)胞,測(cè)定相關(guān)基因的表達(dá)。結(jié)果如下:與對(duì)照組相比,豬和雞回腸滅活總菌顯著提高家兔隱窩細(xì)胞的TLR2、TLR3和TLR4的表達(dá)(P0.05);家兔回腸總菌能顯著提高TLR4的表達(dá)(P0.01)。各處理組對(duì)TLR9均無顯著影響(P0.05)。三種總菌均顯著提高了促炎性因子TNF-α和IL-6的表達(dá)(P0.05)。雞回腸總菌對(duì)抗炎性因子IL-10的表達(dá)刺激作用比其他組顯著(P0.05),且豬和雞回腸總菌提高了IFN-β的表達(dá)(P0.05),豬回腸總菌比家兔回腸總菌有增加IL-10表達(dá)的趨勢(shì)(P0.1),但家兔回腸總菌對(duì)抗炎因子IL-10的表達(dá)沒有促進(jìn)作用(P0.05),甚至有降低IFN-β表達(dá)的趨勢(shì)(P0.1)。(2)用實(shí)驗(yàn)室前期分離的兔源植物乳桿菌L.p2、干酪乳桿菌L.a1和屎腸球菌E.f2,以及購(gòu)買的大腸桿菌E.coli K88滅活后對(duì)仔兔隱窩細(xì)胞進(jìn)行刺激6 h后收集細(xì)胞,測(cè)定相關(guān)基因的表達(dá)。結(jié)果如下:三株乳酸菌顯著提高了TLR2、TLR3和IFN-β的表達(dá)(P0.05)。大腸桿菌E.coli K88顯著提高了TLR4表達(dá)(P0.05)。植物乳酸菌L.p2、屎腸球菌E.f2和大腸桿菌E.coli K88組均顯著提高隱窩細(xì)胞TNF-α的表達(dá)(P0.05)。各處理均顯著提高了IL-6的表達(dá)(P0.05)。植物乳桿菌L.p2和屎腸球菌E.f2顯著提高了IL-10的表達(dá)(P0.05),E.coli K88組IL10的表達(dá)顯著低于對(duì)照組(P0.05)。三株乳酸菌中植物乳桿菌對(duì)促炎性因子的刺激作用更強(qiáng)。3.試驗(yàn)三用豬、雞和家兔回腸總菌及兔源乳酸菌的滅活菌刺激小鼠骨髓源樹突狀細(xì)胞系DC2.4細(xì)胞,結(jié)果表明:(1)DC2.4細(xì)胞受到滅活L.p2刺激后,TLR2、TLR4、TNF-α和IL-10表達(dá)隨著刺激時(shí)間增加呈現(xiàn)先升高后降低的規(guī)律,在2 h前后表達(dá)量較高。(2)豬、雞和家兔回腸總菌刺激DC2.4細(xì)胞2 h后,豬和雞回腸菌比家兔回腸菌顯著提高TLR3表達(dá)(P0.05)。豬和雞回腸菌顯著提高了TLR2和TLR4的表達(dá)(P0.05),家兔回腸菌有提高TLR2的趨勢(shì)(P0.1),但對(duì)TLR3無顯著影響(P0.05)。各處理組對(duì)TLR9表達(dá)無顯著影響(P0.05),均顯著提高了TNF-α及IL-10的表達(dá)(P0.05),但家兔回腸總菌對(duì)TNF-α表達(dá)刺激作用顯著高于豬和雞回腸總菌。雞回腸總菌顯著提高了IL-6和IFN-β的表達(dá)(P0.05),豬回腸總菌有提高IL-6和IFN-β基因表達(dá)的趨勢(shì)(P0.1),但是家兔回腸總菌對(duì)IFN-β的表達(dá)無明顯的影響(P0.05)和有提高IL-6基因表達(dá)的趨勢(shì)(P0.1)。(3)在乳酸菌刺激DC2.4細(xì)胞2 h的試驗(yàn)中,與對(duì)照組相比,L.p2、L.a1和E.f2顯著提高TLR2的表達(dá)(P0.05)。L.a1和E.f2顯著提高了TLR3的表達(dá)(P0.05)。E.coli K88顯著提高了TLR4和TNF-α的表達(dá)(P0.05)。L.p2和E.f2顯著提高了TNF-α基因的表達(dá)(P0.05)。相比對(duì)照組,L.a1和E.f2有增加IFN-β的趨勢(shì)(P0.1)。E.f2和E.coli K88顯著提高了IL-10基因表達(dá)(P0.05)。綜上所述,家兔回腸菌群第一優(yōu)勢(shì)菌門是擬桿菌門,豬和雞第一優(yōu)勢(shì)菌門為厚壁菌門,且家兔回腸乳酸菌遠(yuǎn)遠(yuǎn)低于豬和雞;與豬和雞回腸總菌相比,家兔回腸總菌更容易使隱窩和DC2.4細(xì)胞處于炎癥狀態(tài);滅活乳酸菌能夠引起細(xì)胞免疫反應(yīng)中抗炎性和促炎性因子的同步表達(dá),有利于維持機(jī)體的穩(wěn)態(tài);綜合促炎因子和抗炎因子表達(dá)水平,干酪乳桿菌和屎腸球菌比植物乳桿菌更有利于維持隱窩的炎性穩(wěn)態(tài),但對(duì)DC2.4細(xì)胞的刺激試驗(yàn)沒有獲得相同的結(jié)論。
[Abstract]:The intestinal tract of the rabbit is susceptible to infection, which leads to severe inflammation and subsequent high mortality. The bacteria in the intestinal tract, including the metabolites of the symbiotic bacteria and the body components, can induce intestinal inflammation. The study confirms that the Gram-negative bacteria are more prone to inflammation than the positive bacteria. The main bacteria in each segment of the rabbit were gram-negative bacilli, and the proportion of gram-positive Lactobacillus in the total bacteria was very low. In this paper, we compared the induction of the cultured rabbit's recess cells and the DC inflammatory reaction in the healthy adult pig, chicken and rabbit, and studied several strains of lactic acid bacteria that had been screened in our laboratory. The inflammatory regulation of recess cells and dendritic cells in the intestinal epithelium can be used to investigate whether the unique intestinal flora of the rabbit can cause inflammation in the intestinal tract more easily, and whether the lactic acid bacteria can alleviate the inflammatory reaction, and whether there are differences in the remission of different lactic acid bacteria and the difference of the inflammatory response of two different cell types to the antigen. It provides a theoretical basis for the screening of probiotics in rabbits, improving immunity and reducing the occurrence of enteritis in rabbits. The main contents and results are as follows: 1. the contents of black pigs in the healthy 6 month old Guanzhong, 42 day old Cobo broilers and 60 day old Rex rabbits were collected, and the contents of the ileum in 3 individual animals of each animal were collected. DNA and Illumina HiSeq sequencing were used to analyze the composition of the total bacteria in the ileum of pigs, chickens and rabbits. The results showed that the first dominant bacteria gate of the ileum was 89.32%, the percentage of the total flora was 89.32%, and the best phylum of the pig and chicken ileum was 80.79% and 88.50%. in the ileum, respectively. The abundance of lactobacilli was 19.19%, 81.19% and 0.72%.Beta diversity index showed that the composition of the pig and the chicken ileum group was close, and the difference between the rabbit and the two was two (1), which was two (1) of the experiment 1 pig, chicken and rabbit ileum inactivated by the cultured rabbit recess cells, the blank control group added PBS, the thermal inactivated total bacteria treated hidden bacteria. After 6 h, the cells were collected to determine the expression of related genes. The results were as follows: compared with the control group, the total bacteria of the pig and chicken ileum inactivated significantly increased the expression of TLR2, TLR3 and TLR4 in the recess cells of the rabbit (P0.05), and the total intestinal bacteria in the rabbit could significantly increase the expression of TLR4 (P0.01). There was no significant effect on TLR9 in each group (P0.05). The three kinds of total bacteria were significantly raised. The expression of pro-inflammatory factor TNF- A and IL-6 (P0.05). The expression of total bacteria in the ileum of chicken was significantly higher than that of the other groups (P0.05), and the expression of IFN- beta in pig and chicken ileum increased (P0.05), and the total intestinal bacteria in pig ileum had a tendency to increase the expression of IL-10 (P0.1) than that of the total ileum in rabbits (P0.1), but the total intestinal bacteria in the rabbits were against the cause of inflammation. The expression of subIL-10 was not promoted (P0.05), and even decreased the trend of IFN- beta expression (P0.1). (2) the cells were collected after 6 h stimulation of Lactobacillus plantarum L.p2, Lactobacillus casei L.a1 and Enterococcus faecium E.f2, and the purchased Escherichia coli E.coli K88 inactivated in the early laboratory, and the related genes were detected. The results were as follows: three strains of lactic acid bacteria significantly increased the expression of TLR2, TLR3 and IFN- beta (P0.05). E.coli K88 in Escherichia coli significantly increased the expression of TLR4 (P0.05). The expression of the plant Lactococcus bacteria L.p2, the E.f2 of Enterococcus faecium and the E.coli K88 group of Escherichia coli were significantly increased. 0.05). Lactobacillus plantarum L.p2 and Enterococcus faecium E.f2 significantly increased the expression of IL-10 (P0.05), and the expression of IL10 in E.coli K88 group was significantly lower than that of the control group (P0.05). The stimulation of Lactobacillus plantarum in three strains of Lactobacillus plantarum stronger.3. test three pigs, chicken and rabbit ileum total bacteria and rabbit source of lactic acid bacteria to stimulate mouse bone marrow The result of the source dendritic cell line DC2.4 cells showed that (1) the expression of TLR2, TLR4, TNF- alpha and IL-10 expression increased first and then decreased with the increase of stimulation time. (2) the expression of TLR2, TLR4, TNF- alpha and IL-10 was higher before and after the 2 h. (2) pigs, chickens and rabbit ileum bacteria stimulated DC2.4 cell 2 h, pigs and chicken ileum were more significant than those of rabbit ileum bacteria The expression of TLR3 (P0.05) was enhanced. The expression of TLR2 and TLR4 was significantly increased in pigs and chicken ileum (P0.05), and in rabbit ileum had a tendency to increase TLR2 (P0.1), but there was no significant effect on TLR3 (P0.05). The expression of TNF- alpha and the expression of TLR9 had no significant effect on the expression of TLR9 (P0.05). The expression of IL-6 and IFN- beta was significantly increased in chicken ileum (P0.05), and the trend of IL-6 and IFN- beta gene expression increased (P0.1) in pig ileum total bacteria (P0.1), but there was no obvious effect on the expression of IFN- beta (P0.05) and the trend of improving the expression of IL-6 gene (P0.1) in the total intestinal bacteria of the rabbit (3) in Lactobacillus spines. Compared with the control group, L.p2, L.a1 and E.f2 significantly increased the expression of TLR2 (P0.05).L.a1 and E.f2 significantly increased the expression of TLR3 (P0.05), compared with the control group, and the expression of TLR3 (P0.05) was significantly increased by L.p2, L.a1 and E.f2. The trend (P0.1).E.f2 and E.coli K88 significantly increased the IL-10 gene expression (P0.05). To sum up, the first dominant bacteria gate of the rabbit ileum was the pseudo bacilli gate, the first dominant bacteria gate of the pig and chicken was the thick wall bacteria gate, and the rabbit ileum lactobacillus was far lower than the pig and chicken; compared with the total bacteria of the pig and chicken ileum, the rabbit ileum was more likely to make the recess and DC. The 2.4 cells are in the inflammatory state; inactivating lactic acid bacteria can cause the synchronous expression of anti-inflammatory and proinflammatory factors in the cellular immune response, which is beneficial to maintain the homeostasis of the body; the expression level of pro-inflammatory and anti-inflammatory factors, Lactobacillus casei and Enterococcus faecium are more favorable for maintaining the inflammatory homeostasis of the recess than the Lactobacillus plantarum, but the DC2.4 is fine. The stimulation test of the cell did not obtain the same conclusion.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.291

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐帥;林奕岑;周夢(mèng)佳;倪學(xué)勤;曾東;曾燕;;基于高通量測(cè)定肉雞回腸微生物多樣性及PICRUSt基因預(yù)測(cè)分析[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2016年08期

2 沈雪梅;李晶;張剛;崔宏曉;劉麗慧;姚軍虎;徐秀容;;改良低溫螯合法分離家兔小腸絨毛和隱窩細(xì)胞及分離效果鑒定[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2016年05期

3 許宇靜;張煜坤;沈雪梅;李晶;徐秀容;;采用Illumina MiSeq測(cè)序技術(shù)分析斷奶幼兔盲腸微生物群落的多樣性[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2015年09期

4 張慧;焦志軍;毛朝明;蔣茜;仝佳;王勝軍;許化溪;;林可霉素對(duì)樹突狀細(xì)胞系DC2.4免疫功能影響的初步研究[J];細(xì)胞與分子免疫學(xué)雜志;2011年07期

5 劉雪美;欒淑莉;溫建新;;健康家兔腸道中乳酸菌的分離鑒定[J];青島農(nóng)業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2011年01期

6 高博;楊曉農(nóng);于學(xué)輝;羅薇;黃河;;家兔GAPDH基因?qū)崟r(shí)熒光定量RT-PCR方法的建立[J];中國(guó)畜牧獸醫(yī);2010年01期

7 李桂軍;樓永良;;脆弱擬桿菌腸毒素致病機(jī)制研究進(jìn)展[J];中國(guó)人獸共患病學(xué)報(bào);2009年01期

8 陶可;王永才;童曉莉;周勤飛;譚智忠;熊宜強(qiáng);;兔胃腸道內(nèi)微生物區(qū)系的特點(diǎn)及疫疾的調(diào)控[J];飼料研究;2007年11期

9 吳建忠;杜冰;馮定遠(yuǎn);;乳酸芽孢桿菌制劑對(duì)AA肉雞生產(chǎn)性能的影響[J];中國(guó)飼料;2007年16期

10 金晶;彭穎;李曉波;;快速提取腸道微生物基因組DNA的方法[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2007年01期

相關(guān)博士學(xué)位論文 前4條

1 郭雙雙;木聚糖酶與益生菌緩解腸道炎癥的作用及其機(jī)制[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

2 王寧;腸上皮隱窩細(xì)胞培養(yǎng)體系的創(chuàng)建及利用其體外模擬腫瘤發(fā)生的研究[D];第三軍醫(yī)大學(xué);2014年

3 任大勇;益生乳酸桿菌的黏附及免疫調(diào)節(jié)作用研究[D];吉林大學(xué);2013年

4 張錦華;豬源乳酸桿菌的篩選及其對(duì)仔豬腸道黏膜免疫影響的研究[D];南京農(nóng)業(yè)大學(xué);2011年

相關(guān)碩士學(xué)位論文 前7條

1 沈雪梅;兔源干酪乳桿菌對(duì)仔兔小腸潘氏細(xì)胞分泌功能的影響[D];西北農(nóng)林科技大學(xué);2016年

2 李晶;高黏附性兔源乳酸菌篩選及其對(duì)哺乳仔兔腸道菌群和黏蛋白表達(dá)的影響[D];西北農(nóng)林科技大學(xué);2016年

3 劉暢;旋毛蟲ES抗原及HSP70對(duì)樹突狀細(xì)胞免疫調(diào)節(jié)的研究[D];東北農(nóng)業(yè)大學(xué);2014年

4 張煜坤;脆弱擬桿菌與枯草芽孢桿菌對(duì)仔兔腸道微生物區(qū)系及免疫的影響[D];西北農(nóng)林科技大學(xué);2014年

5 牛坤偉;自身免疫調(diào)節(jié)因子對(duì)DC2.4細(xì)胞表型及功能影響的研究[D];吉林大學(xué);2014年

6 李亞力;乳酸菌對(duì)幼齡獺兔生產(chǎn)性能與免疫性能的影響[D];西北農(nóng)林科技大學(xué);2013年

7 朱克華;酸化飲水對(duì)斷奶幼兔生長(zhǎng)性能、消化酶活性及腸道環(huán)境的影響[D];西北農(nóng)林科技大學(xué);2012年

，

本文編號(hào)：1872812