小鼠肠道菌群失衡模型建立及菌群失衡对肠道Toll样受体的影响

发布时间：2018-07-18 14:44

【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。
[Abstract]:Purpose 1. Based on the changes of 14 dominant microflora in intestinal tract, a mouse model of microflora imbalance with different degrees was established by using antibiotic interference. To study the effect of intestinal microflora imbalance on Toll-like receptor signal transduction pathway in mouse intestinal mucosa epithelium, and to investigate the molecular response of microecological flora imbalance and intestinal immune barrier and its role and mechanism in the occurrence and development of infectious diseases. To provide new ideas and strategies for the prevention and treatment of infectious diseases. Methods: 1. The model of intestinal flora imbalance in mice was established by intragastric administration of ceftriaxone sodium. The intestinal flora of cecum contents was analyzed with the dose of 5g/kg/d for 8 days, and the model of microflora imbalance was established by using the contents of cecum for 8 days, and the intestinal flora was analyzed with the dose of 8g/kg/d at the final concentration for 8 days. A model of severe bacterial imbalance was established. 2. The changes of TLR9, TLR4, TLR2, NF- 魏 Bp65 were detected by RT-PCR in normal group, mild disequilibrium group and severe dysbacteriosis group by EDTA oscillatory method, and the changes of NF- 魏 Bp65 were detected by immunohistochemistry and RT-PCR. Results: 1. By means of medium improvement and culture identification method, the genus Bifidobacterium, Lactobacillus, Bacteroides, Eubacteribacterium, Enterococcus, Streptococcus, Clostridium, Veronella, Digestive Staphylococcus, Macrococcus were established. Methods of culture counting and analysis of stable intestinal dominant flora of Enterobacter, Staphylococcus, Saccharomyces and Mycetes. 2. Using antibiotic ceftriaxone sodium 5 g / kg / d for 8 consecutive days, the dominant flora decreased significantly, and a model of slight microbial imbalance was established, and when ceftriaxone sodium 8 g / kg / d for 8 consecutive days, the number of dominant bacteria decreased to almost zero. A mouse model of severe bacterial imbalance was established. Compared with normal mice, the expression of TLR9 and TLR4 were decreased, and the changes of TLR4 and TLR2 in severe group were more obvious than those in mild group. 4. Compared with the normal group, the level of NF- 魏 B p65 in the unbalance group was higher than that in the normal group, and the serious imbalance was significantly higher than that in the mild group. Conclusion: using antibiotic ceftriaxone sodium can establish a stable model of mild and severe bacterial imbalance in mice, and the changes of Toll-like receptors in intestinal mucosal epithelium are affected by the changes of bacterial flora. The expression of TLR9 decreased and the expressions of TLR2 and TLR4 increased. The expression of NF- 魏 B p65 increased.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R-332;R574

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