【摘要】： 目的:1.以腸道十四種優(yōu)勢菌群變化情況為依據(jù),利用抗生素干擾建立不同程度的菌群失衡小鼠模型。2.研究腸道菌群失衡對小鼠腸粘膜上皮Toll樣受體信號轉導通路的影響,探明微生態(tài)菌群失衡與腸道免疫屏障的分子應答及其在感染性疾病發(fā)生、發(fā)展中的作用和機理,為感染性疾病的防治提供新思路和新策略。 方法: 1.采用抗生素頭孢曲松鈉灌胃的方式建立小鼠腸道菌群失衡模型。以終濃度5g/kg/d的劑量,連續(xù)8天,取盲腸內(nèi)容物進行腸道菌群分析,建立輕度菌群失衡模型;以終濃度8g/kg/d的劑量,連續(xù)8天,進行腸道菌群分析,建立重度菌群失衡模型。 2.利用EDTA振蕩法提取正常組、輕度菌群失衡組、重度菌群失衡小鼠腸粘膜上皮細胞;RT-PCR方法檢測TLR9、TLR4、TLR2的變化;免疫組織化學方法和RT-PCR方法檢測NF-κBp65的變化。 結果: 1.通過培養(yǎng)基改良和培養(yǎng)鑒定方法,建立了腸道雙歧桿菌屬、乳酸桿菌屬、類桿菌屬、優(yōu)桿菌屬、腸球菌屬、鏈球菌屬、梭桿菌屬、韋榮球菌屬、消化球菌屬、巨球菌屬、腸桿菌屬、葡萄球菌屬、酵母菌屬、霉菌屬十四種腸道優(yōu)勢菌群穩(wěn)定的培養(yǎng)計數(shù)分析方法。 2.利用抗生素頭孢曲松鈉5g/kg/d,連續(xù)8天灌胃,優(yōu)勢菌群顯著下降,建立輕度菌群失衡模型;以頭孢曲松鈉8g/kg/d,連續(xù)8天灌胃,優(yōu)勢菌群的數(shù)量幾乎降至零,建立重度菌群失衡小鼠模型。 3.菌群失衡小鼠與正常組小鼠相比較,TLR9表達均呈下降趨勢,TLR4和TLR2均呈升高趨勢,重度菌群失衡較輕度菌群失衡變化明顯。 4.菌群失衡小鼠與正常組小鼠相比較,NF-κBp65均呈升高趨勢,且重度失衡較輕度失衡升高明顯。 結論:利用抗生素頭孢曲松鈉能建立穩(wěn)定的輕度菌群失衡模型和重度菌群失衡小鼠模型;腸粘膜上皮Toll樣受體變化受菌群變化的影響,菌群失衡促使TLR9表達呈下降趨勢;TLR2和TLR4表達呈升高趨勢;NF-κBp65表達呈升高趨勢。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R-332;R574

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本文編號：2132231