【作者】 刘洋；

【导师】 唐立；

【作者基本信息】 大连医科大学 ， 病原生物学， 2009， 硕士

【摘要】 目的:1.以肠道十四种优势菌群变化情况为依据,利用抗生素干扰建立不同程度的菌群失衡小鼠模型。2.研究肠道菌群失衡对小鼠肠粘膜上皮Toll样受体信号转导通路的影响,探明微生态菌群失衡与肠道免疫屏障的分子应答及其在感染性疾病发生、发展中的作用和机理,为感染性疾病的防治提供新思路和新策略。方法:1.采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型。以终浓度5g/kg/d的剂量,连续8天,取盲肠内容物进行肠道菌群分析,建立轻度菌群失衡模型;以终浓度8g/kg/d的剂量,连续8天,进行肠道菌群分析,建立重度菌群失衡模型。2.利用EDTA振荡法提取正常组、轻度菌群失衡组、重度菌群失衡小鼠肠粘膜上皮细胞;RT-PCR方法检测TLR9、TLR4、TLR2的变化;免疫组织化学方法和RT-PCR方法检测NF-κBp65的变化。结果:1.通过培养基改良和培养鉴定方法,建立了肠道双歧杆菌属、乳酸杆菌属、类杆菌属、优杆菌属、肠球菌属、链球菌属、梭杆菌属、韦荣球菌属、消化球菌属、巨球菌属、肠杆菌属、葡萄球菌属、酵母菌属、霉菌属十四种肠道优势菌群稳定的培养计数分析方法。2.利用抗生素头孢曲松钠5g/kg/d,连续8天灌胃,优势菌群显著下降,建立轻度菌群失衡模型;以头孢曲松钠8g/kg/d,连续8天灌胃,优势菌群的数量几乎降至零,建立重度菌群失衡小鼠模型。3.菌群失衡小鼠与正常组小鼠相比较,TLR9表达均呈下降趋势,TLR4和TLR2均呈升高趋势,重度菌群失衡较轻度菌群失衡变化明显。4.菌群失衡小鼠与正常组小鼠相比较,NF-κBp65均呈升高趋势,且重度失衡较轻度失衡升高明显。结论:利用抗生素头孢曲松钠能建立稳定的轻度菌群失衡模型和重度菌群失衡小鼠模型;肠粘膜上皮Toll样受体变化受菌群变化的影响,菌群失衡促使TLR9表达呈下降趋势;TLR2和TLR4表达呈升高趋势;NF-κBp65表达呈升高趋势。更多还原

【Abstract】 Objective: 1.Utilize antibiotics to effect intestinal microbiology of Balb/c mice, to establish mice model for dysbiosis of intestinal microflora based on intestinal predominant microbial population changes; 2.Toll-like receptor-mediated signaling of intestinal epithelium cells(IECs) influenced by alteration of intestinal flora, to investigate the molecular interaction of commensal bacteria with the intestinal epithelium and the mucosal immune system;to supply infectious diseases therapy with new thinking and strategy.Methods:1.We administer Balb/c mice with different doses of ceftriaxone sodium, establishing mice model for dysbiosis of Intestinal flora. Utilize 5g/kg/d doses, 8 days to establish light micro dysbiosis; meanwhile utilize 8g/kg/d doses, 8 days to establish heavy micro dysbiosis.2.In normal group, light micro dysbiosis and heavy micro dysbiosis animals intestinal epithelium cells were isolated Using an EDTA-viration method; Toll-like recptor 2, 4 and 9 mRNA were determined by RT-PCR; NF-κBp65 was determined by RT-PCR and immunohistochemistry.Results:1.Establish consummate analytical method of fourteen kinds of intestinal dominant microbe including Bacillus bifidus, Lactobacillus, Bateroides, Eubacterium, Enterococus, Streptococcus, Fusobacterium, Veillonella, Escherichia, Staphylococcus, yeast and so on.2.Using 5g/kg/d doses of ceftriaxone sodium to intragastic administrate Balb/c mice, we discovered dominant microbe decreased obviously and establish light micro dysbiosis; meanwhile utilize 8g/kg/d doses, dominant microbe decreased to zero, establishing heavy micro dysbiosis.3.Compared with normal mice, the levels of TLR9 was lower and TLR4 and TLR2 were higher of mRNA in dysbacteria mice, moreover the heavy-dysbacteria mice were higher than light-dysbacteria mice.4.Compared with normal mice, the levels of NF-κBp65 was up-regulation of both mRNA and protein in dysbacteria mice, moreover the heavy-dysbacteria mice were higher than light-dysbacteria mice.Conclusion: Use different doses of ceftriaxone sodium to intragastic administrate Balb/c mice, we establish mice model for heavy and light dysbiosis of intestinal microflora; compared with normal mice, the levels of TLR9 was lower and TLR4 and TLR2 were higher of mRNA in dysbacteria mice; NF-κBp65 was up-regulation of both mRNA and protein.更多还原