【摘要】：益生菌的應用已持續(xù)數十年,雖然其益生作用機理尚不清楚,但在臨床上對消化道疾病的顯著療效已經得到充分的證實,對機體免疫功能的保護和增強作用愈加受到關注。一般認為益生菌在臨床治療腸道感染性腹瀉的分子和細胞機制主要涉及三個方面:(1)益生菌可產生抗菌物質并競爭性與病原性微生物黏附于腸上皮細胞來阻止病原體在腸道的黏附和定植;(2)益生菌可提高病毒感染時腸上皮細胞的存活率和保護腸道的正常生理結構并刺激保護性免疫反應來調節(jié)機體免疫;(3)益生菌可通過調節(jié)腸道微生態(tài)平衡來保護和抵御病原微生物的侵襲。本研究主要從分子水平、細胞水平以及動物體內試驗三個方面闡述豬腸道益生乳酸菌對傳染性胃腸炎病毒(TGEV)和豬流行性腹瀉病毒(PEDV)的抑制作用和免疫調節(jié)作用,獲得了以下結果:(1)分離篩選出在豬睪丸細胞(ST細胞)上對TGEV具有抑制作用的3株豬源腸道益生菌Y091231、Y091221和Y224031,經16S rDNA鑒定分別為唾液乳桿菌(Lactobacillus saliarius),約氏乳桿菌(Lactobacillus johnsonii)和糞腸球菌(Enterococcus faecalis)。(2)篩選菌對感染TGEV的ST細胞具有保護作用。3株益生菌及其代謝產物接入順序不同,對病毒的抑制率也不同,Y091231菌懸液在預先接入的情況下可以達到最大抑制率(65%),而Y091221和Y224031則在與病毒共同接入時表現出較高的抑制率。通過半定量RT-PCR檢測結果可知,3株益生菌及其代謝產物均可以降低TGEV在ST細胞內的增殖,Y091231菌懸液在各處理組中具有穩(wěn)定的較高的病毒抑制力。益生菌在體外抗TGEV作用中,可以通過上調IL-8和IL-12、下調IL-4和INF-α這4種細胞因子在細胞培養(yǎng)上清中的表達量,從而刺激細胞免疫,起到抵御病毒入侵的作用。(3)分離鑒定PEDV-WN株,構建動物模型。從臨床病料中分離得到PEDV,然后對毒株進行RT-PCR鑒定及M、S基因序列分析,與CV777株對比分析,PEDV-WN株的M基因具有高度保守性,可以作為檢測PEDV的靶基因;S基因變異較大,S蛋白內分的核心和表位(CEO,499-638aa)共有9個氨基酸位點發(fā)生了突變。將PEDV-WN株回歸試驗動物,成功構建了典型PEDV感染動物模型。(4)唾液乳桿菌(Lactobacillus saliarius)對PEDV-WN株具有抑制作用,對病毒感染的仔豬具有保護作用。該菌株的預防性干預和治療性干預均能提高仔豬存活率,提高仔豬生長性能,減輕病毒對腸道組織的病理損傷,并能降低病毒在機體內的增殖。益生菌可以調節(jié)機體IL-4、IL-8、IL-12、INF-α4種細胞因子和MHCⅡ的表達量,從而刺激機體的細胞免疫和體液免疫,起到抗病毒的作用。(5)PEDV感染嚴重破壞了正常的腸道菌群結構和菌種豐度,唾液乳桿菌的干預可以保護腸道微生態(tài)保持健康的平衡狀態(tài),并能迅速修復失調的腸道微生態(tài)。通過對糞便菌群的16S rDNA高通量測序的方法,對PEDV感染和益生菌干預的不同試驗組的腸道菌群的變化規(guī)律和差異性進行了對比分析。
[Abstract]:The application of probiotics has lasted for decades. Although the mechanism of probiotics is not clear, the remarkable effect of probiotics on digestive tract diseases has been fully confirmed, and the protection and enhancement of immune function have been paid more and more attention. It is generally believed that the molecular and cellular mechanisms of probiotics in the treatment of intestinal infectious diarrhea are mainly related to three aspects: (1) probiotics can produce antimicrobial substances and be adhered to intestinal epithelial cells by competitive and pathogenic microorganisms to prevent pathogens. (2) probiotics can improve the survival rate of intestinal epithelial cells and protect the normal physiological structure of the intestine and stimulate the protective immune response to regulate the immune system; (3) probiotics can regulate the immune system by regulating the intestinal tract. Tao microecological balance to protect and protect against the invasion of pathogenic microorganisms. The inhibitory and immunomodulatory effects of porcine enteric probiotic lactic acid bacteria on infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) were discussed from three aspects: molecular level, cell level and animal test in vivo. The following results were obtained: (1) three strains of porcine enteroprobiotics Y091231, Y091221 and Y224031 were isolated and screened to inhibit TGEV in porcine testicular cells (St cells). They were identified as Lactobacillus saliva (Lactobacillus saliarius), (Lactobacillus johnsonii) and fecal intestine by 16s rDNA, respectively. Streptococcus (Enterococcus faecalis). (_ 2) showed a protective effect on St cells infected with TGEV. 3 strains of probiotics and their metabolites were inserted in different sequences. The inhibition rate of Y091231 strain was also different, and that of Y091221 and Y224031 was higher than that of Y091221 and Y224031. The results of semi-quantitative RT-PCR showed that three strains of probiotics and their metabolites could reduce the proliferation of TGEV in St cells. Probiotics can up-regulate the expression of IL-8 and IL-12 and down-regulate the expression of IL-4 and INF- 偽 in the supernatant of cell culture, thus stimulating cellular immunity and resisting virus invasion. (3) isolation and identification of PEDV-WN strain. Build animal model. PEDVV was isolated from clinical materials. The M gene of PEDV-WN strain was highly conserved by RT-PCR identification and sequence analysis of MNs gene of PEDV-WN strain, and compared with CV777 strain, the M gene of PEDV-WN strain was highly conserved. A total of 9 amino acid loci were mutated at the core and epitope (CEO 499-638aa), which can be used as the target gene for PEDV detection. The typical animal model of PEDV infection was successfully constructed by regression of PEDV-WN strain. (4) Lactobacillus saliva (Lactobacillus saliarius) inhibited PEDV-WN strain and protected piglets infected by the virus. The preventive and therapeutic intervention of the strain can improve the survival rate of piglets, improve the growth performance of piglets, alleviate the pathological damage caused by the virus to the intestinal tissue, and reduce the proliferation of the virus in the body. Probiotics can regulate the expression of IL-4, IL-8, IL-12, INF- 偽 and MHC 鈪，

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