【作者】 修晶辉；

【导师】 魏萍；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 本实验从健康的雏鸡和仔猪盲肠中分离鉴定出三株益生菌,采用MTT和细胞病变方法,分别在鸡胚成纤维细胞(CEF)和PK-15细胞模型上初步评价了三株益生菌对TGEV和IBV抑制率的影响,探讨益生菌的抗病毒作用。实验中首先利用革兰氏染色,生化试验,16Sr RNA,抑菌试验等方法分离鉴定出了三株益生菌,分别命名为:L1—德氏乳杆菌德氏亚种(Lactobacillus.delbrueckiisubsp.delbrueckii)、L2—德氏乳杆菌乳亚种(Lactobacillus.delbrueckii subsplactis)和L3—德氏乳杆菌保加利亚亚种( Lactobacillus delbrueckii subsp. bulgaricus)。利用MTT法测定益生菌处理后的细胞活性,经统计分析得出益生菌对细胞的最大无毒浓度为L1株1011 cfu/ml,L2株1012 cfu/ml,L3株1012 cfu/ml,益生菌培养上清1:2的稀释液,益生菌灭活菌体和破碎菌体成分在实验浓度均未显现细胞毒害现象。根据上述实验结果开展下列益生菌的抗病毒作用研究。实验分组:第1组:益生菌与上述2种细胞预先分别孵育后再攻毒;第2组:2种细胞感染病毒后再接入益生菌;第3组:益生菌与病毒同时接入细胞;第4组:益生菌对照;第5组:病毒对照;第6组:正常细胞对照。实验结果:以MTT法测定益生菌和病毒(TGEV和IBV)处理后的细胞活性,通过公式计算病毒抑制率。结果表明,无论是在PK-15细胞上,还是在CEF细胞上,益生菌成分和不同加入方式均可显著地提高益生菌对TGEV和IBV的抑制率(P﹤0.05),并且这种作用具有量效关系和时效关系。益生菌-细胞-病毒相互作用后的细胞病变结果显示,益生菌及其培养上清与TGEV和IBV同时接入细胞可以极显著的阻碍TGEV和IBV引发的细胞病变,推测其可能具有直接的杀灭病毒作用。在实验中,我们分别研究了益生菌活菌、培养上清、灭活菌体和破碎菌体成分的抗病毒作用,结果表明,益生菌的各种形式均具有一定的抗病毒作用,但在效果上存在一定的差异,原因可能是其抗病毒作用的机制不同。益生菌与细胞作用后再感染病毒,对TGEV和IBV抑制率升高的结果反映出了益生菌对TGEV和IBV吸附细胞的阻断作用;从益生菌与病毒同时接入细胞后降低病毒对细胞侵害的现象,可以看出益生菌可能对病毒具有直接破坏作用;细胞感染病毒后再接入益生菌对TGEV和IBV抑制率极低的现象说明,病毒感染后益生菌再很难发挥作用。综上所述,在体外益生菌对TGEV和IBV细胞病变影响的实验中,所分离的三株益生菌及其培养上清都极显著地阻碍了TGEV和IBV细胞病变程度,但各菌种之间存在程度不同的差异;这一结果说明益生菌可能具有直接破坏病毒的作用,并且具有菌种特异性。初步确定这三株益生菌具有抗病毒作用。益生菌菌株可以直接破坏TGEV和IBV,又可以阻断病毒对细胞的感染、抑制病毒在细胞内的增殖。更多还原

【Abstract】 In the experiments, Bolting and identify three strains probiotics from cecum of healthy chicken and young swine, apply MTT and cytopathic effect(CPE) method in vitro CEF and PK-15 cell preliminary assessment the effect of the inhibition rate of probiotics to TGEV and IBV, then to draw the antiviral activity of three probiotics and to analyze the possible mechanism.In experiments, first to utilize Gram staining, biochemistry essay, 16Sr RNA , bacteriostasis essay and so on bolting and identify three strains probiotics is Lactobacillus.delbrueckiisubsp. delbrueckii(L1);Lactobacillus.delbrueckiisubsplactis(L2);Lactobacillus.delbrueckiisubsp.Bulgaricus (L3) .We determined the cell viability after treated by probiotics using MTT and analyzed the largest non-toxic concentration of 1011cfu/ml Lactobacillus casei, 1012cfu/ml of Lactobacillus acidophilus and 1:2 diluted probiotics metabolites, inactivated probiotics and broken products in the experimental concentrations had not appear cell toxicity phenomena.we selected the highest sensitivity antibiotics(gentamicin) on the three probiotics by sensitivity test.According to the experimental results, we had the anti-virus research of probiotics in cells. Experimental groups as follows: group 1: Pre-treatment of cells with probiotics; Group 2: Cells infected with the virus and then added probiotics; Group 3: Co-incubation of probiotics and NDV; Group 4: Probiotics control; Group 5: Virus control; Group 6: Cells control.The results showed:We determined the cell activity after probiotics and TGEV and IBV treated by MTT method,and alculated the viral inhibition rate by formula .The results showed that, whether in the PK-15 cell, or in the CEF, probiotics componentcould and different add methods significantly rise the inhibition rate of TGEV and IBV, and had a relationship with concentrations and time.Cytopathic effect(CPE) of probiotics-cell- virus interaction showed that probiotics and their metabolites added to cells with TGEV and IBV at the same time can reduce the titer of TGEV and IBV significantly, and it indicated that it may have a direct role in killing virus .In the experiment, we studied the antiviral ability of probiotics, metabolites, inactivated probiotics and broken components, results showed that the probiotics in various forms all have some antiviral effect, but there are some differences may be due to the different mechanisms of its antiviral activity .The result of TGEV and IBV inhibition rate rose when pre-treatment of cells with probiotics reflected that the probiotics may block the adsorption of TGEV and IBV on cells; from the phenomenon probiotics and virus added to cells at the same time reduced the harm of virus on cells,we can see that probiotics can play a direct role of damage the virus structure; the phenomenon of lowest inhibition rate of TGEV and IBV when cells infected with TGEV and IBV before probiotics added showed that after the cells infected the probiotics will hard to have effect on virus.In summary, in the experiment of probiotics on the TGEV and IBV titer in vitro, three probiotics separated and its metabolites were significantly reduced the titer of TGEV and IBV, but there are some differences between strains; the three probiotics separated and their metabolites did not affect the titer of TGEV and IBV, the results indicated that probiotics may have a direct role in destruction of the virus and have strain-specific probiotics not only has a direct role in destruction of the virus, but also can block the virus infected the cells and inhibit the proliferation of virus in cells.更多还原