本文選題：解淀粉芽孢桿菌 + 沙門氏菌��； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：益生菌可調(diào)節(jié)動(dòng)物腸道微生態(tài)平衡和免疫功能,從而改善動(dòng)物生產(chǎn)性能。芽孢桿菌制劑因其較好的抗逆性而得到廣泛應(yīng)用。本課題組前期已篩選獲得一株可顯著促進(jìn)仔豬生長(zhǎng)和改善腸粘膜免疫功能的解淀粉芽孢桿菌SC06(BaSC06)。本論文研究了 BaSC06對(duì)小鼠抗沙門氏菌(Salmonella enterica serovar Typhimurium,ST)感染和腸道巨噬細(xì)胞極化的影響及機(jī)理,旨在探明BaSC06對(duì)體內(nèi)巨噬細(xì)胞極化的調(diào)控及其與抗ST感染的相關(guān)性主要研究?jī)?nèi)容和結(jié)果如下:(一)飼喂BaSC06對(duì)小鼠抗ST感染能力和腸道巨噬細(xì)胞極化的影響雄性C57BL/6小鼠隨機(jī)分為四組,分別為Control組,BaSC06組,ST組和BaSC06+ST組。Control組和ST組小鼠飼喂普通飼糧,BaSC06組和BaSC06+ST組小鼠飼喂含有BaSC06的飼糧,連續(xù)飼喂8周。然后ST組和BaSC06+ST組小鼠灌服ST,3天后處死取樣。研究結(jié)果表明:(1)飼喂BaSC06顯著緩解由ST引起的小鼠體重下降、盲腸萎縮、回盲腸和結(jié)腸粘膜損傷及腸道炎癥;(2)飼喂BaSC06可顯著降低ST在小鼠腸系膜淋巴結(jié)(Mesenteric lymph node,MLN)和脾臟中的移位,ST感染前后,BaSC06對(duì)派伊爾結(jié)(Peyer's patches,PP)、MLN和脾臟中的CD4:CD8比值均無(wú)顯著影響,表明BaSC06不能調(diào)節(jié)T細(xì)胞分化。飼喂BaSC06對(duì)小鼠血清細(xì)胞因子IL-1β、IL-4、、IL-12、IL-10和IL-13無(wú)顯著影響,顯著增加IFN-γ含量;但可顯著降低由ST感染引起的促炎因子IL-1β和IL-12的增加,促進(jìn)抑炎因子IL-10的表達(dá)。以上結(jié)果表明,飼喂BaSC06可有效降低ST感染引起的系統(tǒng)性損傷和炎癥反應(yīng);(3)飼喂BaSC06可明顯降低ST感染引起的盲腸巨噬細(xì)胞浸潤(rùn),同時(shí)誘導(dǎo)盲腸巨噬細(xì)胞向M1型和M2型極化,顯著增加ST感染小鼠MLN中M1和M2型巨噬細(xì)胞數(shù)量。以上結(jié)果提示,飼喂BaSC06可誘導(dǎo)腸道巨噬細(xì)胞向M1型和M2型極化,有效緩解ST引起的小鼠腸道和全身感染。(二)BaSC06對(duì)巨噬細(xì)胞極化的影響以BaSC06處理小鼠巨噬細(xì)胞系Raw264.7,結(jié)果發(fā)現(xiàn):(1)BaSC06與巨噬細(xì)胞共培養(yǎng)6h后顯著增強(qiáng)巨噬細(xì)胞的吞噬殺菌功能;(2)基因表達(dá)和Western Blot檢測(cè)表明BaSC06促進(jìn)巨噬細(xì)胞向M1型極化;(3)流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)ST感染可促進(jìn)巨噬細(xì)胞向M2型極化,BaSC06與ST共同作用24h后促進(jìn)巨噬細(xì)胞M1型極化,抑制M2型極化。腹腔注射BaSC06(106CFU/只)或ST(105CFU/只),12h后麻醉處死小鼠并分離腹腔巨噬細(xì)胞進(jìn)行流式細(xì)胞術(shù)分析,結(jié)果表明:(1)ST誘導(dǎo)腹腔巨噬細(xì)胞向M2型極化,抑制M1型極化。BaSC06可誘導(dǎo)腹腔巨噬細(xì)胞向M1型極化,對(duì)M2型極化無(wú)顯著影響;(2)過(guò)繼回輸經(jīng)BaSC06誘導(dǎo)的原代巨噬細(xì)胞顯著降低了脾臟的ST載菌量。以上結(jié)果表明BaSC06通過(guò)誘導(dǎo)巨噬細(xì)胞M1型極化增強(qiáng)其對(duì)ST的殺菌活性。通過(guò)構(gòu)建抗生素小鼠模型可排除腸道菌群對(duì)結(jié)果的干擾,直接驗(yàn)證BaSC06對(duì)腸道巨噬細(xì)胞的極化的影響。每只小鼠灌喂20mg鏈霉素后灌服ST和BaSC06。結(jié)果表明,與抗生素處理組相比,ST感染誘導(dǎo)腸道巨噬細(xì)胞向M2型極化,對(duì)M1型巨噬細(xì)胞無(wú)顯著影響,BaSC06促進(jìn)腸道巨噬細(xì)胞向M1型極化,抑制ST誘導(dǎo)的M2型極化。以上結(jié)果表明,ST感染誘導(dǎo)巨噬細(xì)胞向M2型極化,BaSC06可直接誘導(dǎo)巨噬細(xì)胞向M1型極化,逆轉(zhuǎn)ST誘導(dǎo)的M2型極化,促進(jìn)細(xì)胞和機(jī)體清除ST。(三)解淀粉芽孢桿菌BaSC06調(diào)節(jié)的腸道菌群對(duì)沙門氏菌感染及巨噬細(xì)胞極化的影響飼喂BaSC06同時(shí)誘導(dǎo)小鼠腸道內(nèi)巨噬細(xì)胞向M1型和M2型極化,BaSC06可直接誘導(dǎo)巨噬細(xì)胞向M1型極化,鑒于益生菌可調(diào)節(jié)腸道菌群發(fā)揮其益生作用,故推測(cè)BaSC06可能通過(guò)調(diào)節(jié)腸道菌群促進(jìn)巨噬細(xì)胞向M2型極化。小鼠腸道菌群的分析結(jié)果表明:(1)飼喂BaSC06對(duì)健康小鼠的腸道菌群多樣性無(wú)顯著影響,但BaSC06可顯著緩解ST感染導(dǎo)致的菌群多樣性的降低。ST感染可降低疣微菌門(Verruccomicrobia)的比例,增加擬桿菌門(Bacterodetes)的相對(duì)豐度;而飼喂BaSC06顯著升高疣微菌門(Verrucomicrobia)的比例,降低擬桿菌門(Bacterodetes)的相對(duì)豐度,表明BaSC06可緩解ST引起的菌群失衡。將Control組和飼喂BaSC06組小鼠的糞便菌群移植給抗生素處理后的小鼠,檢測(cè)盲腸炎癥和巨噬細(xì)胞極化,結(jié)果表明:(1)移植BaSC06組小鼠的糞便菌群后能夠緩解ST引起的盲腸中性粒細(xì)胞浸潤(rùn),緩解盲腸炎癥;(2)BaSC06組小鼠糞便菌群能夠促進(jìn)盲腸巨噬細(xì)胞向M2型極化。以上結(jié)果表明BaSC06可通過(guò)調(diào)節(jié)腸道菌群促進(jìn)巨噬細(xì)胞向M2型極化,維持腸道穩(wěn)態(tài),緩解ST感染引起的炎癥反應(yīng)。綜上所述,飼喂BaSC06可直接促進(jìn)巨噬細(xì)胞M1型極化,增加其對(duì)ST的殺傷功能,同時(shí)BaSC06調(diào)節(jié)的腸道菌群能夠促進(jìn)巨噬細(xì)胞向M2型極化,維持腸道的微生態(tài)穩(wěn)態(tài)和炎癥平衡,飼喂BaSC06后誘導(dǎo)的腸道M1型和M2型巨噬細(xì)胞共同發(fā)揮抵抗ST感染的效果。
[Abstract]:Probiotics can regulate the intestinal microecological balance and immune function of animals, thus improving animal production performance. Bacillus spore preparation has been widely used for its good resistance to adversity. In our group, a strain of Bacillus starches SC06 (BaSC06), which could significantly promote the growth of piglets and improve intestinal mucosal immune function, was screened in our group. The effects and mechanisms of BaSC06 on Salmonella enterica serovar Typhimurium (ST) infection and intestinal macrophage polarization in mice were studied. The purpose of this study was to explore the main contents and results of the regulation of BaSC06 on the polarization of macrophages in vivo and its correlation with the anti ST infection. (1) the ability of feeding BaSC06 to resist ST infection in mice was fed. The male C57BL/6 mice were randomly divided into four groups: group Control, group BaSC06, group ST and BaSC06+ST group.Control and ST group of mice fed with common diet, BaSC06 and BaSC06+ST mice fed with BaSC06 diet and fed for 8 weeks. Then ST group and BaSC06+ST group mice were sacrificed in 3 days. The results were as follows: (1) feeding BaSC06 significantly alleviated the loss of weight in mice, cecum atrophy, cecum and colonic mucosa injury and intestinal inflammation caused by ST; (2) feeding BaSC06 significantly reduced the shift of ST in the mesenteric lymph nodes (Mesenteric, node, MLN) and the spleen in mice, and BaSC06 was the Peyer's P (Peyer's P) before and after ST infection. Atches, PP), the ratio of CD4:CD8 in MLN and spleen had no significant influence, indicating that BaSC06 did not regulate the differentiation of T cells. Feeding BaSC06 had no significant effect on the serum cytokines of IL-1 beta, IL-4, IL-12, IL-10 and IL-13. The above results show that BaSC06 can effectively reduce the systemic damage and inflammatory response caused by ST infection. (3) feeding BaSC06 can significantly reduce the infiltration of cecum macrophage caused by ST infection, and induce the cecum macrophage to M1 and M2 polarization, significantly increasing the number of M1 and M2 macrophages in ST infected mice MLN. The results suggested that feeding BaSC06 could induce M1 and M2 type polarization of intestinal macrophages, effectively alleviating intestinal and systemic infection in mice caused by ST. (two) the effect of BaSC06 on the polarization of macrophages was treated with BaSC06 to treat the mouse macrophage system Raw264.7. The results were as follows: (1) BaSC06 and macrophage co culture 6h significantly enhanced macrophage. Phagocytosis and bactericidal function; (2) gene expression and Western Blot detection showed that BaSC06 promoted macrophage to M1 polarization; (3) flow cytometry detected that ST infection could promote M2 type polarization of macrophages. BaSC06 and ST promote M1 polarization of macrophages and inhibit M2 polarization. Only after 12h anesthesia, the mice were killed and the peritoneal macrophages were isolated and analyzed by flow cytometry. The results showed: (1) ST induced M2 polarization in peritoneal macrophages, and M1 polarization.BaSC06 could induce M1 polarization in peritoneal macrophages, and there was no significant effect on M2 polarization; (2) the primary macrophage induced by adoptive return transfusion was significantly reduced. The results showed that BaSC06 could enhance its bactericidal activity to ST by inducing M1 polarization of macrophages. By constructing an antibiotic mouse model, the effects of intestinal flora on the results were excluded, and the effect of BaSC06 on the polarization of intestinal macrophages was directly verified. Each mouse was fed with 20mg streptomycin and filled with ST and BaSC06. after perfusion of streptomycin. The results showed that compared with the antibiotic treatment group, ST infection induced M2 polarization in the intestinal macrophages and had no significant effect on M1 type macrophages. BaSC06 promoted the M1 polarization of intestinal macrophages and inhibited the M2 polarization induced by ST. The results showed that ST infection induced macrophage to M2 polarization, and BaSC06 can directly induce macrophage to M1 type. Polarization, reversing ST induced M2 polarization, promoting cell and organism clearance of ST. (three) solution of Bacillus amyloid BaSC06 regulating the effect of intestinal microflora on Salmonella infection and macrophage polarization, feeding BaSC06 and inducing macrophage to M1 and M2 polarization in the intestinal tract of mice, and BaSC06 can directly induce macrophage to M1 polarization. Probiotics can regulate intestinal flora to play its probiotic effect, so it is presumed that BaSC06 may promote M2 polarization by regulating intestinal microflora. The analysis of intestinal microflora in mice showed that: (1) feeding BaSC06 had no significant effect on the diversity of intestinal flora in healthy mice, but BaSC06 could significantly alleviate the diversity of bacterial flora caused by ST infection. The reduction of.ST infection could reduce the proportion of Verruccomicrobia and increase the relative abundance of Bacterodetes, while feeding BaSC06 significantly increased the proportion of verruca microphylum (Verrucomicrobia) and reduced the relative abundance of the bacteribacillus gate (Bacterodetes), and indicated that BaSC06 could alleviate the imbalance of the bacterial flora caused by ST. Control group and BaSC06 were fed to BaSC06. The fecal flora of the mice was transplanted to the mice treated with antibiotics to detect the cecum inflammation and macrophage polarization. The results were as follows: (1) the fecal flora of the BaSC06 group can relieve the infiltration of the cecum neutrophils caused by ST and alleviate the cecum inflammation; (2) the fecal flora of group BaSC06 can promote the cecum macrophage to M2 type. The above results show that BaSC06 can promote M2 polarization by regulating intestinal microflora, maintain intestinal homeostasis and alleviate the inflammatory response caused by ST infection. In summary, feeding BaSC06 can directly promote M1 polarization of macrophages and increase the killing function of macrophages on ST, while BaSC06 regulated intestinal flora can promote macrophage to M2 Type polarization, maintain the balance of intestinal microecology and inflammation, and induce intestinal type M1 and M2 macrophages to resist ST infection after feeding BaSC06.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S816

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