β-防御素及Toll样受体参与分泌性中耳炎发病的体内研究

发布时间：2018-05-09 04:17

本文选题：β-防御素 + 分泌性中耳炎　；参考：《复旦大学》2010年硕士论文

【摘要】： 第一部分β-防御素在分泌性中耳炎大鼠咽鼓管鼓室的表达目的检测rBD-1、rBD-2 mRNA在分泌性中耳炎大鼠咽鼓管鼓室粘膜中的表达,初步探讨β-防御素在分泌性中耳炎发病机制中的作用。方法排除中耳感染的清洁级SD大鼠48只,随机分为4组,每组12只。前3组36只行颈部切口经右侧听泡注入脂多糖溶液(lmg/ml)30μL,造模后分别于1、3、7天断头取咽鼓管鼓室粘膜；对照组12只大鼠右侧听泡注入生理盐水30μL,左侧听泡作为空白对照组,与造模3天组同时取咽鼓管鼓室粘膜。逆转录聚合酶链反应(RT-PCR)法检测咽鼓管鼓室粘膜rBD-1、rBD-2在mRNA水平的表达,Image J软件分析电泳图并进行半定量。结果正常SD大鼠咽鼓管鼓室粘膜存在rBD-1、rBD-2 mRNA的表达,rBD-1 mRNA的相对表达量较rBD-2 mRNA强(P0.01)。造模后1、3、7天,rBD-1 mRNA表达变化不明显,rBD-2 mRNA表达在造模后1、3天增加明显(P0.01),第7天渐回复到正常水平。结论在SD大鼠,rBD-1可能参与正常咽鼓管鼓室的防御功能,造模后rBD-2的表达增加可能与病原体入侵后的清除相关。第二部分TLR-2、TLR-4在分泌性中耳炎小鼠咽鼓管鼓室的表达目的用PAF诱导BALB/c小鼠分泌性中耳炎模型,检测造模前后TLR-2、TLR-4在咽鼓管鼓室粘膜的表达,探讨PAF、TLR与分泌性中耳炎发病之间的关系。方法 SPF级BALB/c小鼠50只,排除中耳感染,随机分为5组,每组10只。前4组经双侧鼓膜注入PAF 5μL(1μg)制作分泌性中耳炎小鼠模型,造模组8只小鼠分别于1、3、7、10天断头取咽鼓管鼓室粘膜；对照组8只小鼠右侧听泡注入生理盐水5μL,左侧听泡作为正常对照组,与造模7天组同时取咽鼓管鼓室粘膜。每组另2只小鼠取听泡固定并脱钙后制作冰冻切片,行HE染色观察造模后组织学变化。逆转录聚合酶链反应(RT-PCR),蛋白质印迹(western-blot)检测小鼠咽鼓管鼓室粘膜TLR-2、TLR-4的表达；免疫荧光(冰冻切片)确认TLR-2、TLR-4表达的具体部位。结果 PAF 1μg经鼓膜注射诱导的BALB/c小鼠分泌性中耳炎,炎症可持续10天。正常BALB/c小鼠咽鼓管鼓室粘膜存在TLR-2、TLR-4 mRNA的表达,两者相对表达量比较差异不显著；PAF诱导后两者表达均增强,造模前后的表达差异有显著性(P0.01);western-blot证实炎症粘膜中TLR-2、TLR-4在蛋白水平的表达高于正常粘膜(P0.01)；免疫荧光显示TLR-2、TLR-4广泛表达于鼓室粘膜,在炎症鼓室粘膜中表达有增强(P0.01),咽鼓管粘膜中主要为TLR-2绿色荧光,鼓膜及中耳腔炎症细胞表面主要为TLR-4红色荧光。结论 PAF在中耳粘膜诱导TLR-2、TLR-4表达上调,PAF诱导的分泌性中耳炎小鼠模型可用以进一步研究PAF、TLR在分泌性中耳炎发病中的具体作用及相互联系。
[Abstract]:Part I expression of 尾 -defensin in eustachian tympanum of rats with secretory otitis media Purpose To investigate the role of 尾 -defensin in the pathogenesis of secretory otitis media by detecting the expression of rBD-1rBD-2 mRNA in the tympanic mucosa of eustachian tube in rats with secretory otitis media. Method Forty-eight clean SD rats excluding middle ear infection were randomly divided into 4 groups with 12 rats in each group. In the first three groups, 36 rats were injected with lipopolysaccharide (LPS) solution through right auditory bubble through the neck incision, and 30 渭 L of lipopolysaccharide solution was injected into the right auditory bubble. The mucosa of eustachian tube tympanum was cut off at 1, 3 and 7 days after modeling, while the right auditory bubble of 12 rats in the control group was injected with normal saline 30 渭 L, and the left auditory bubble was taken as the blank control group. The mucosa of tympanic cavity of eustachian tube was taken at the same time as the model group for 3 days. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of rBD-1 and rBD-2 in eustachian tube tympanic mucosa at mRNA level. Result The expression of rBD-1rBD-2 mRNA in eustachian tube tympanic mucosa of SD rats was stronger than that of rBD-2 mRNA. The expression of rBD-1 mRNA did not change significantly on the 7th day after modeling. The expression of rBD-2 mRNA increased significantly on the 1st and 3rd day after modeling, and gradually returned to the normal level on the 7th day. Conclusion In SD rats, rBD-1 may be involved in the defense function of normal eustachian tube tympanum, and the increased expression of rBD-2 after modeling may be related to the clearance of pathogens after invasion. Part II expression of TLR-2TLR-4 in eustachian tube tympanum of mice with secretory otitis media Purpose A model of secretory otitis media in BALB/c mice induced by PAF was used to detect the expression of TLR-2 and TLR-4 in the tympanic mucosa of eustachian tube before and after the establishment of the model, and to explore the relationship between the expression of TLR-4 and the pathogenesis of secretory otitis media. Method SPF grade BALB/c mice were randomly divided into 5 groups with 10 rats in each group. The model of secretory otitis media was established in the first four groups by injecting PAF 5 渭 L 1 渭 g through bilateral tympanic membrane. The mucous membrane of the tympanic cavity of the eustachian tube was taken from 8 mice in the model group after 10 days. 8 mice in the control group were injected with normal saline 5 渭 L on the right side, and the mucosa of the tympanic cavity of the eustachian tube was taken from the left auditory bubble as the control group and the model group for 7 days. The other 2 mice in each group were fixed with auditory vesicles and decalcified to make frozen sections. The histological changes were observed by HE staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot (Western blot) were used to detect the expression of TLR-2 and TLR-4 in mouse eustachian tube tympanic mucosa, and the specific site of TLR-2TLR-4 expression was confirmed by immunofluorescence (frozen section). Result PAF 1 渭 g was injected into tympanic membrane to induce secretory otitis media in BALB/c mice. The inflammation lasted for 10 days. The expression of TLR-2 and TLR-4 mRNA in eustachian tympanic mucosa of normal BALB/c mice was not significantly different. The expression of TLR-2 and TLR-4 in inflammatory mucosa was higher than that in normal mucosa (P 0.01), and immunofluorescence showed that TLR-2 and TLR-4 were widely expressed in tympanic mucosa. In the mucosa of the inflammatory tympanum, the expression of P0.01G was enhanced, the green fluorescence of TLR-2 was mainly found in the mucosa of eustachian tube, and the TLR-4 red fluorescence was mainly found on the surface of the tympanic membrane and middle ear cavity. Conclusion The mouse model of secretory otitis media induced by PAF up-regulated the expression of TLR-2 and TLR-4 in middle ear mucosa could be used to further study the specific role and relationship of PAF-TLR in the pathogenesis of secretory otitis media.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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