發(fā)布時(shí)間：2018-04-27 06:08

  本文選題：屎腸球菌 + 豬小腸黏膜��； 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：屎腸球菌是仔豬生產(chǎn)中常用的益生菌,但是其作用機(jī)理尚不清楚。為了更充分地了解屎腸球菌對(duì)仔豬小腸黏膜先天防御屏障功能的影響,本研究用屎腸球菌活菌和熱滅活菌對(duì)新生仔豬進(jìn)行試驗(yàn),并研究它們對(duì)豬腸上皮細(xì)胞的影響。動(dòng)物試驗(yàn)選用窩產(chǎn)仔數(shù)和體重相近、胎次和出生時(shí)間相同的9窩仔豬,隨機(jī)分為3個(gè)組,每組3窩仔豬。從出生開始,對(duì)照組、活菌組和滅活菌組仔豬就分別灌服10%的脫脂乳溶液、含益生屎腸球菌活菌(1×10~8cfu/mL)的10%脫脂乳溶液和含熱滅活屎腸球菌(1×10~8cfu/mL)的10%脫脂乳溶液各10 mL,每天一次,連續(xù)6天,仔豬7日齡開始飼喂教槽料,21日齡斷奶。記錄仔豬出生、7日齡以及21日齡體重,并統(tǒng)計(jì)教槽前和斷奶前的腹瀉率,并在7日齡飼喂教槽料之前,每窩選擇一頭中等體重的仔豬屠宰,采集腸粘膜樣本,分析相關(guān)指標(biāo)。細(xì)胞試驗(yàn)采用豬空腸上皮細(xì)胞系IPEC-J2細(xì)胞,設(shè)5個(gè)處理,每個(gè)處理6個(gè)重復(fù)。細(xì)胞培養(yǎng)在37℃,5%CO_2恒溫培養(yǎng)箱中,在IPEC-J2細(xì)胞生長交匯后,對(duì)照組、LPS組、活菌組、滅活菌組和細(xì)胞壁組分別在無抗無血清培養(yǎng)基中加入PBS、LPS(1 ng/mL)、益生屎腸球菌活菌(1 × 10~8cfu/mL)、熱滅活屎腸球菌(1×10~8cfu/mL)和細(xì)胞壁(100μg/mL),與 IPEC-J2 細(xì)胞共孵育 12 h,分別在1、3、6和12 h收集細(xì)胞培養(yǎng)上清和細(xì)胞裂解液,并測(cè)定相關(guān)指標(biāo)。主要試驗(yàn)結(jié)果如下:1.益生屎腸球菌活菌及其熱滅活菌都不影響哺乳期仔豬的體增重,但都能大幅度降低仔豬的腹瀉率,而且活菌組脾臟指數(shù)下降顯著(p0.05)。益生屎腸球菌活菌顯著上調(diào)空腸和回腸黏膜的Zo-1基因表達(dá)(P0.05),不影響occludin基因表達(dá);但空腸黏膜的occludin和Zo-1基因表達(dá)不受熱滅活菌影響,回腸黏膜的這兩種緊密連接蛋白顯著下調(diào)(p0.05)。Western blot結(jié)果顯示,益生屎腸球菌活菌與熱滅活菌都可提高空腸和回腸黏膜occludin和claudin的蛋白表達(dá)。與IPEC-J2共培養(yǎng)3h內(nèi),屎腸球菌活菌、熱滅活菌以及其細(xì)胞壁都能上調(diào)zo-1、occludin的表達(dá)水平(p0.05),活菌和滅活菌還上調(diào)了共孵育1h的clauaine表達(dá)水平(p0.05),當(dāng)共培養(yǎng)時(shí)間長達(dá)6h以上時(shí),緊密連接蛋白的表達(dá)都下調(diào)(p0.05),而LPS對(duì)這三種緊密連接蛋白的基因表達(dá)即使在共培養(yǎng)3h內(nèi)也基本都是顯著下調(diào)作用(p0.05)。Western blot結(jié)果顯示,在3h內(nèi),屎腸球菌活菌可上調(diào)occludin和claudin的蛋白表達(dá)水平,熱滅活菌和細(xì)胞壁組在lh內(nèi)上調(diào)claudin的蛋白表達(dá)量,3h時(shí)與對(duì)照組相比差異不顯著,6h時(shí)活菌與熱滅活菌的緊密連接蛋白表達(dá)量都趨于下降,而LPS組從lh開始就下調(diào)occludin和claudin的蛋白表達(dá)水平,蛋白表達(dá)與基因表達(dá)基本一致。2.屎腸球菌活菌顯著上調(diào)仔豬空腸黏膜的il-1、il-6、il-8、tnf-α和tgf-βmRNA表達(dá)水平(p0.05),但是顯著下調(diào)仔豬回腸黏膜的il-1、il-6、il-12和tgf-β的表達(dá)水平(p0.05),不影響空腸黏膜的i1-12和回腸黏膜的il-8和tnf-α的表達(dá)量。與活菌不同,熱滅活菌組的空腸黏膜的i l-1、il-6、i1-8、i1-12、tnf-α 和回腸黏膜的 il-6、1l-8、il-12 mRNA表達(dá)水平都顯著下調(diào)(p0.05),而空腸黏膜的tgf-β和回腸黏膜的il-1、tnf-α、tgf-β的表達(dá)量則不變。屎腸球菌活菌和熱滅活菌對(duì)仔豬小腸黏膜細(xì)胞因子分泌的影響也有差別�；罹@著促進(jìn)空腸黏膜和回腸黏膜TGF-β以及回腸黏膜IL-8的分泌(P0.05),不影響空腸黏膜IL-8、空腸和回腸黏膜IL-10和TNF-α的分泌量。而熱滅活菌組的空腸黏膜IL-10和TGF-β的分泌量都明顯增加(p0.05),回腸黏膜IL-8、TNF-α和TGF-β的分泌量卻都顯著下降(p0.05),但是空腸黏膜IL-8、TNF-α和回腸黏膜IL-10的分泌量不受影響。與IPEC-J2共培養(yǎng)lh時(shí),屎腸球菌活菌顯著上調(diào)il-1、il-8、tn-α和tgf-βmRNA的表達(dá)量(p0.05),但il-6和il-12mRNA的表達(dá)水平被顯著抑制(p0.05);熱滅活菌只顯著上調(diào)了 il-6、i1-8和tgf-βmRNA的表達(dá)水平(p0.05),而il-1、il-12和tnf-α都不受影響;細(xì)胞壁僅僅顯著下調(diào)了 i1-8和tnf-α mRNA的表達(dá)量(p0.05),對(duì)il-1、il-6、i1-12和tgf-β不影響。當(dāng)共培養(yǎng)時(shí)間達(dá)到3h時(shí),屎腸球菌活菌則顯著上調(diào)il-1、il-6、il-8和tnf-α mRNA的表達(dá)量(p0.05),熱滅活菌顯著促進(jìn)1l-1、i1-6、il-8和il-12的表達(dá)(p0.05),細(xì)胞壁使得i1-6和il-12 mRNA的表達(dá)量顯著增加(p0.05),但是卻顯著抑制tnf-α的表達(dá)(p0.05)。不管共孵育1h還是3h,屎腸球菌活菌都顯著刺激工PEC-J2細(xì)胞分泌TGF-β,但卻顯著抑制IL-8和IL-10的產(chǎn)生,不影響TNF-α的濃度。而熱滅活菌與IPEC-J2共孵育時(shí),只是顯著增加1h的TGF-β分泌量,但對(duì)3h的沒有影響,并顯著減少共孵育1h和3h的工L-8和IL-10產(chǎn)量,對(duì)TNF-α也沒有影響。3.屎腸球菌活菌組仔豬空腸黏膜的TLR-2和TLR-4以及回腸黏膜的TLR-9基因表達(dá)都顯著上調(diào)(p0.05),但是空腸黏膜的TLR-9以及回腸黏膜TLR-2和TLR-4基因的表達(dá)水平都不受影響;而熱滅活菌組仔豬空腸黏膜和回腸黏膜的TLR-4以及回腸黏膜的TLR-9基因表達(dá)都顯著下調(diào)(p0.05),但是不影響空腸和回腸黏膜TLR-2以及空腸黏膜的TLR-9基因的表達(dá)。與IPEC-J2共培養(yǎng)3h時(shí),屎腸球菌活菌組和細(xì)胞壁組的TLR-2表達(dá)量都顯著增加(p0.05),但TLR-9的表達(dá)不受影響,而熱滅活菌組和LPS組則都顯著減少TLR-9的表達(dá)量(p0.05),但不影響TLR-2的表達(dá);活菌組、熱滅活菌組以及LPS組都促進(jìn)TLR-4的表達(dá)(p0.05),但是細(xì)胞壁卻顯著抑制TLR-4的表達(dá)(p0.05)。結(jié)論:益生屎腸球菌的活菌和熱滅活菌有相似的益生作用(減少仔豬腹瀉),但是它們對(duì)仔豬小腸黏膜先天防御屏障的調(diào)節(jié)作用不完全一樣:活菌增強(qiáng)仔豬小腸黏膜(包括豬腸上皮細(xì)胞)緊密連接蛋白的表達(dá),并活化小腸黏膜(包括豬腸上皮細(xì)胞)的TLR2,4,9,誘導(dǎo)以耐受為主導(dǎo)的免疫應(yīng)答;而熱滅活菌只能促進(jìn)豬腸上皮細(xì)胞表達(dá)緊密連接蛋白和TLR-4,但是也能誘導(dǎo)仔豬小腸黏膜產(chǎn)生耐受為主體的應(yīng)答。
[Abstract]:Enterococcus faecium is a common probiotic in the production of piglets, but its mechanism is still unclear. In order to understand the effect of Enterococcus faecium to the intestinal mucosal defense barrier function of intestinal mucosa of piglets more fully, this study used Enterococcus faecium and thermal inactivated bacteria to test the newborn piglets and study their effects on the pig intestinal epithelial cells. 9 piglets with similar litter size and similar weight, birth weight and birth time were randomly divided into 3 groups, each group of 3 piglets. From birth, the control group, the living bacteria group and the inactivated group piglets were filled with 10% degreased milk solution, including the 10% degreased milk solution of probiotic Enterococcus faecium (1 x 10~8cfu/mL) and the heat inactivated excrement ball. Bacteria (1 * 10~8cfu/mL) of 10% degreased milk solution each 10 mL, once a day for 6 days. The piglets were fed at 7 days of age and were weaned at 21 days of age. The piglets were born, 7 days and 21 days of age were recorded, and the diarrhea rate before and before the slots were recorded. Before feeding the grooves at the age of 7 days, the piglets were slaughtered for each nest of medium weight. The intestinal mucosal samples were used to analyze the related indexes. The cell test used the porcine jejunum epithelial cell line IPEC-J2 cells and set up 5 treatments, each treated with 6 repetitions. Cell culture was at 37, 5%CO_2 constant temperature incubator. After the growth of IPEC-J2 cells, the control group, the LPS group, the live bacteria group and the cell wall group were added to the serum-free medium without anti serum-free medium. PBS, LPS (1 ng/mL), probiotic Enterococcus faecium live bacteria (1 x 10~8cfu/mL), heat inactivated Enterococcus faecium (1 x 10~8cfu/mL) and cell wall (100 mu g/mL), incubating 12 h with IPEC-J2 cells, collecting cell culture supernatant and cell lysate in 1,3,6 and 12 h respectively, and determining the phase index. The main test results are as follows: 1. probiotic Enterococcus faecium live bacteria and its Heat inactivated bacteria did not affect the body weight gain of suckling piglets, but the diarrhea rate of piglets was greatly reduced, and the spleen index of the living bacteria group decreased significantly (P0.05). The Zo-1 gene expression of the jejunum and ileum mucosa was significantly up-regulated by probiotic Enterococcus faecium (P0.05), and the expression of occludin gene was not affected; but the occludin and Zo-1 gene tables of the jejunum mucosa were expressed. The two close connexin of the ileum mucous membrane decreased significantly (P0.05).Western blot results showed that both probiotic Enterococcus and thermo inactivated bacteria could increase the protein expression of occludin and claudin in the jejunum and ileum mucosa. In co culture of 3H, Enterococcus faecium, thermal inactivated bacteria and its cell wall in IPEC-J2 co culture. The expression level of ZO-1, occludin (P0.05), living bacteria and inactivated bacteria also increased the clauaine expression level of CO incubated 1H (P0.05). When the co culture time was longer than 6h, the expression of close connexin was reduced (P0.05), and the gene expression of these three closely connexin proteins was substantially reduced even in co culture 3H. The results of action (P0.05).Western blot showed that in 3h, Enterococcus faecium could increase the protein expression level of occludin and claudin. The protein expression of claudin was up-regulated in LH by hot inactivated bacteria and cell wall groups, and there was no significant difference compared with the control group in 3H. The expression of close connexin protein of both active and thermal inactivated bacteria tended to decrease while 6h, and LPS, while LPS. The protein expression level of occludin and claudin was downregulated from LH, and the expression of protein expression and gene expression was basically consistent with the expression level of IL-1, IL-6, IL-8, tnf- A and tgf- beta mRNA (P0.05) in the intestinal mucosa of piglets, but the expression level of the intestinal mucosa of piglets was significantly reduced, but the expression level of the intestinal mucosa of piglets was significantly reduced. The expression of IL-8 and tnf- alpha in the i1-12 and ileum mucosa of the jejunum mucosa. Different from the living bacteria, the expressions of I L-1, IL-6, i1-8, i1-12, tnf- alpha and the il-6,1l-8 of the ileum mucosa in the heat inactivated bacteria group were significantly down down, while the expression of the intestinal mucosa of the mucosa of the jejunum and the ileum mucosa, the expression of alpha, and beta The effects of Enterococcus faecium and heat inactivated bacteria on the secretion of cytokines in intestinal mucosa of piglets were also different. The active bacteria significantly promoted the secretion of TGF- beta in the jejunum mucosa and ileum mucosa and the secretion of IL-8 in the ileum mucosa (P0.05), and did not affect the secretion of IL-10 and TNF- alpha in the jejunum mucosa IL-8, the jejunum and ileum mucosa, and the jejunum mucosa I in the heat inactivated bacteria group The secretion of L-10 and TGF- beta was significantly increased (P0.05), but the secretion of IL-8, TNF- A and TGF- beta in the ileum mucosa decreased significantly (P0.05), but the secretion of IL-8, TNF- alpha and IL-10 in the ileum mucosa was not affected. The expression level of L-6 and il-12mRNA was significantly inhibited (P0.05), and the expression level of IL-6, i1-8 and tgf- beta mRNA was significantly up-regulated by thermal inactivated bacteria (P0.05), but IL-1, IL-12 and tnf- alpha were not affected. At the time, the Enterococcus faecium increased the expression of IL-1, IL-6, IL-8 and tnf- alpha mRNA (P0.05), and the heat inactivated bacteria significantly promoted the expression of 1L-1, i1-6, IL-8 and IL-12 (P0.05). The bacteria significantly stimulated the secretion of TGF- beta in the PEC-J2 cells, but significantly inhibited the production of IL-8 and IL-10, and did not affect the concentration of TNF- alpha, but when the thermal inactivated bacteria were incubated with IPEC-J2, it only significantly increased the TGF- beta secretion of 1H, but had no effect on 3h, and significantly reduced the L-8 and yield of 1H and 3h, and did not affect the excrement. The expression of TLR-2 and TLR-4 in the jejunum mucosa of enterococci and the expression of TLR-9 gene in the ileum mucosa were significantly up (P0.05), but the TLR-9 of the jejunum mucosa and the expression level of TLR-2 and TLR-4 genes in the ileum mucosa were not affected; and the TLR-9 gene of the jejunum mucosa and ileum mucosa of the piglet and the ileum mucosa in the heat inactivated bacteria group The expression of the TLR-9 gene in the jejunum and ileum mucosa TLR-2 and the jejunum mucosa was not affected by the expression of the expression of TLR-9 in the jejunum and ileum mucosa. The expression of TLR-2 in the Enterococcus and cell wall groups increased significantly (P0.05) when 3H was co cultured with IPEC-J2 (P0.05), but the expression of TLR-9 was not affected, while the thermally inactivated and LPS groups significantly reduced the table of TLR-9. P0.05, but did not affect the expression of TLR-2; living bacteria, heat inactivated and LPS groups all promote the expression of TLR-4 (P0.05), but the cell wall significantly inhibits the expression of TLR-4 (P0.05). Conclusion: probiotics of Enterococcus faecium and thermal inactivated bacteria have similar probiotics (reducing piglet diarrhea), but they are innate against the small intestinal mucosa of piglets. The regulatory effect of the Royal barrier is not exactly the same: the living bacteria enhance the expression of close connexin in the small intestinal mucosa of piglets (including pig intestinal epithelial cells), and activate the TLR2,4,9 of the small intestinal mucosa (including the pig intestinal epithelial cells), and induce the tolerant immune response, while the thermally inactivated bacteria can only promote the expression of close connexin and TLR- in the pig intestinal epithelial cells. 4, but it also induced the tolerance of piglets to the main body.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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