益生屎肠球菌活菌和热灭活菌调节仔猪小肠黏膜紧密连接蛋白、细胞因子以及相关的Toll样受体的研究

发布时间：2018-04-27 06:08

本文选题：屎肠球菌 + 猪小肠黏膜　；参考：《广西大学》2017年硕士论文

【摘要】：屎肠球菌是仔猪生产中常用的益生菌,但是其作用机理尚不清楚。为了更充分地了解屎肠球菌对仔猪小肠黏膜先天防御屏障功能的影响,本研究用屎肠球菌活菌和热灭活菌对新生仔猪进行试验,并研究它们对猪肠上皮细胞的影响。动物试验选用窝产仔数和体重相近、胎次和出生时间相同的9窝仔猪,随机分为3个组,每组3窝仔猪。从出生开始,对照组、活菌组和灭活菌组仔猪就分别灌服10%的脱脂乳溶液、含益生屎肠球菌活菌(1×10~8cfu/mL)的10%脱脂乳溶液和含热灭活屎肠球菌(1×10~8cfu/mL)的10%脱脂乳溶液各10 mL,每天一次,连续6天,仔猪7日龄开始饲喂教槽料,21日龄断奶。记录仔猪出生、7日龄以及21日龄体重,并统计教槽前和断奶前的腹泻率,并在7日龄饲喂教槽料之前,每窝选择一头中等体重的仔猪屠宰,采集肠粘膜样本,分析相关指标。细胞试验采用猪空肠上皮细胞系IPEC-J2细胞,设5个处理,每个处理6个重复。细胞培养在37℃,5%CO_2恒温培养箱中,在IPEC-J2细胞生长交汇后,对照组、LPS组、活菌组、灭活菌组和细胞壁组分别在无抗无血清培养基中加入PBS、LPS(1 ng/mL)、益生屎肠球菌活菌(1 × 10~8cfu/mL)、热灭活屎肠球菌(1×10~8cfu/mL)和细胞壁(100μg/mL),与 IPEC-J2 细胞共孵育 12 h,分别在1、3、6和12 h收集细胞培养上清和细胞裂解液,并测定相关指标。主要试验结果如下:1.益生屎肠球菌活菌及其热灭活菌都不影响哺乳期仔猪的体增重,但都能大幅度降低仔猪的腹泻率,而且活菌组脾脏指数下降显著(p0.05)。益生屎肠球菌活菌显著上调空肠和回肠黏膜的Zo-1基因表达(P0.05),不影响occludin基因表达;但空肠黏膜的occludin和Zo-1基因表达不受热灭活菌影响,回肠黏膜的这两种紧密连接蛋白显著下调(p0.05)。Western blot结果显示,益生屎肠球菌活菌与热灭活菌都可提高空肠和回肠黏膜occludin和claudin的蛋白表达。与IPEC-J2共培养3h内,屎肠球菌活菌、热灭活菌以及其细胞壁都能上调zo-1、occludin的表达水平(p0.05),活菌和灭活菌还上调了共孵育1h的clauaine表达水平(p0.05),当共培养时间长达6h以上时,紧密连接蛋白的表达都下调(p0.05),而LPS对这三种紧密连接蛋白的基因表达即使在共培养3h内也基本都是显著下调作用(p0.05)。Western blot结果显示,在3h内,屎肠球菌活菌可上调occludin和claudin的蛋白表达水平,热灭活菌和细胞壁组在lh内上调claudin的蛋白表达量,3h时与对照组相比差异不显著,6h时活菌与热灭活菌的紧密连接蛋白表达量都趋于下降,而LPS组从lh开始就下调occludin和claudin的蛋白表达水平,蛋白表达与基因表达基本一致。2.屎肠球菌活菌显著上调仔猪空肠黏膜的il-1、il-6、il-8、tnf-α和tgf-βmRNA表达水平(p0.05),但是显著下调仔猪回肠黏膜的il-1、il-6、il-12和tgf-β的表达水平(p0.05),不影响空肠黏膜的i1-12和回肠黏膜的il-8和tnf-α的表达量。与活菌不同,热灭活菌组的空肠黏膜的i l-1、il-6、i1-8、i1-12、tnf-α 和回肠黏膜的 il-6、1l-8、il-12 mRNA表达水平都显著下调(p0.05),而空肠黏膜的tgf-β和回肠黏膜的il-1、tnf-α、tgf-β的表达量则不变。屎肠球菌活菌和热灭活菌对仔猪小肠黏膜细胞因子分泌的影响也有差别。活菌显著促进空肠黏膜和回肠黏膜TGF-β以及回肠黏膜IL-8的分泌(P0.05),不影响空肠黏膜IL-8、空肠和回肠黏膜IL-10和TNF-α的分泌量。而热灭活菌组的空肠黏膜IL-10和TGF-β的分泌量都明显增加(p0.05),回肠黏膜IL-8、TNF-α和TGF-β的分泌量却都显著下降(p0.05),但是空肠黏膜IL-8、TNF-α和回肠黏膜IL-10的分泌量不受影响。与IPEC-J2共培养lh时,屎肠球菌活菌显著上调il-1、il-8、tn-α和tgf-βmRNA的表达量(p0.05),但il-6和il-12mRNA的表达水平被显著抑制(p0.05);热灭活菌只显著上调了 il-6、i1-8和tgf-βmRNA的表达水平(p0.05),而il-1、il-12和tnf-α都不受影响;细胞壁仅仅显著下调了 i1-8和tnf-α mRNA的表达量(p0.05),对il-1、il-6、i1-12和tgf-β不影响。当共培养时间达到3h时,屎肠球菌活菌则显著上调il-1、il-6、il-8和tnf-α mRNA的表达量(p0.05),热灭活菌显著促进1l-1、i1-6、il-8和il-12的表达(p0.05),细胞壁使得i1-6和il-12 mRNA的表达量显著增加(p0.05),但是却显著抑制tnf-α的表达(p0.05)。不管共孵育1h还是3h,屎肠球菌活菌都显著刺激工PEC-J2细胞分泌TGF-β,但却显著抑制IL-8和IL-10的产生,不影响TNF-α的浓度。而热灭活菌与IPEC-J2共孵育时,只是显著增加1h的TGF-β分泌量,但对3h的没有影响,并显著减少共孵育1h和3h的工L-8和IL-10产量,对TNF-α也没有影响。3.屎肠球菌活菌组仔猪空肠黏膜的TLR-2和TLR-4以及回肠黏膜的TLR-9基因表达都显著上调(p0.05),但是空肠黏膜的TLR-9以及回肠黏膜TLR-2和TLR-4基因的表达水平都不受影响;而热灭活菌组仔猪空肠黏膜和回肠黏膜的TLR-4以及回肠黏膜的TLR-9基因表达都显著下调(p0.05),但是不影响空肠和回肠黏膜TLR-2以及空肠黏膜的TLR-9基因的表达。与IPEC-J2共培养3h时,屎肠球菌活菌组和细胞壁组的TLR-2表达量都显著增加(p0.05),但TLR-9的表达不受影响,而热灭活菌组和LPS组则都显著减少TLR-9的表达量(p0.05),但不影响TLR-2的表达;活菌组、热灭活菌组以及LPS组都促进TLR-4的表达(p0.05),但是细胞壁却显著抑制TLR-4的表达(p0.05)。结论:益生屎肠球菌的活菌和热灭活菌有相似的益生作用(减少仔猪腹泻),但是它们对仔猪小肠黏膜先天防御屏障的调节作用不完全一样:活菌增强仔猪小肠黏膜(包括猪肠上皮细胞)紧密连接蛋白的表达,并活化小肠黏膜(包括猪肠上皮细胞)的TLR2,4,9,诱导以耐受为主导的免疫应答;而热灭活菌只能促进猪肠上皮细胞表达紧密连接蛋白和TLR-4,但是也能诱导仔猪小肠黏膜产生耐受为主体的应答。
[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.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

【参考文献】