TNF-α与CD44在SD大鼠分泌性中耳炎的表达及意义

发布时间：2018-04-01 08:35

本文选题：分泌性中耳炎　切入点：鼓室积液　出处：《泸州医学院》2011年硕士论文

【摘要】：目的：分泌性中耳炎(Otitis media with effusion, OME)是以鼓室积液为特征的慢性非化脓性炎症,其发病机制目前尚不明确,通常认为与咽鼓管机械性阻塞、感染、免疫反应有关。本实验通过建立OME大鼠模型,研究TNF-α与CD44在SD大鼠听泡粘膜上皮中的表达变化,探讨TNF-α与CD44在OME发生、发展中所起的作用。方法：成年健康SD大鼠40只,设立正常对照组、手术对照(PBS)组、OME模型组三个实验组。正常对照组选SD大鼠4只,不作任何处理,直接取听泡表面粘膜及听泡灌洗液检测；手术对照(PBS)组选用SD大鼠16只分别在中耳腔内注射磷酸缓冲液(PBS),按2天、4天、7天、15天四个不同时间点处死SD大鼠后取听泡灌洗液和听泡粘膜组织检测；OME模型组在中耳腔注射脂多糖(LPS)后,与手术对照(PBS)组相对应,按四个不同时间点取听泡粘膜组织及大鼠听泡灌洗液行检测(各时间段均多选入一只SD大鼠,以防出现造模过程中动物死亡所致实验动物数量不够之情况)。听泡灌洗液运用ELISA法检测各组中肿瘤坏死因子(TNF-α)浓度。听泡粘膜组织通过固定、包埋制成切片,运用HE染色法查看各组大鼠听听泡粘膜组织细胞的形态学改变,采用免疫组化SP法检测听泡粘膜中粘附分子CD44及TNF-α在不同实验组中的阳性表达,用Image-Pro Plus 6.0多媒体彩色病理图像分析系统进行分析,统计学分析得出实验结论。结果：1、采用ELISA法对正常对照组听泡灌洗液中的TNF-α浓度进行检测,其平均浓度为：91.831ng/ml。手术对照(PBS)组听泡灌洗液中平均TNF-α浓度在术后2天、4天、7天、15天时为148.858ng/ml、169.898ng/ml、135.753ng/ml、101.830ng/ml。OME实验组,听泡渗液中的TNF-α平均浓度分别为201.020ng/ml、235.774ng/ml、230.681ng/ml、116.958ng/ml。OME实验组灌洗液的TNF-α浓度在受LPS影响后的七天左右最高,其后逐渐减轻,在15天时,炎症因子降至最低点。以a=0.05为检验标准,做组间配对t检验,OME实验组与正常对照组和手术对照(PBS)组比较有明显差异(P0.05)。提示在OME大鼠模型,TNF-α的浓度在OME实验组显著高于对照组。2、HE染色切片可见,正常对照组听泡粘膜无炎症改变；手术对照(PBS)组术后2天、4天、7天、15天均未见听泡粘膜上皮细胞有明显改变,仅粘膜下有少量炎症细胞浸润；OME实验组术后两天可见听泡粘膜水肿,炎性细胞浸润,其中有大量中性粒细胞。术后四天组见毛细血管扩张,炎性细胞浸润增强,以大量中性粒细胞为主。术后七天组可见上皮下间隙增宽,更多的炎性细胞浸润,仍以大量中性粒细胞为主。术后15天组出现成纤维细胞增生,新生血管增多,并可见到单核巨噬细胞浸润,中耳粘膜明显增厚的改变。3、免疫组化SP染色,正常对照组TNF-α和CD44均在粘膜上皮及上皮下结缔组织仅少量表达。手术对照(PBS)组TNF-α和CD44染色阳性的细胞表达部位与正常对照组无变化,且四个时间点之间表达强度和阳性细胞数无明显差异(P0.05),无统计学意义。OME实验组听泡粘膜上皮细胞和粘膜下结缔组织中均有TNF-α和CD44阳性表达,尤其在杯状细胞及粘膜下混合腺强烈表达,其术后15天强烈表达。经Image-Pro Plus 6.0检测双侧听泡粘膜阳性细胞灰度值,使用SPSS13.0采用组间t检验、相关性分析。OME实验组与手术对照(PBS)组和正常对照组TNF-α和CD44阳性表达经统计学分析发现：OME实验组与对照组间两种因子的表达强度有明显差异(P0.05)。通过相关性分析,发现OME实验组内,TNF-α和CD44表达明显相关。手术组不同时间段TNF-α和CD44阳性表达无明显差异(P0.05),无统计学意义。结论：1、在OME大鼠模型急性期,鼓室积液的中TNF-α浓度与炎症的严重程度密切相关。2、TNF-α介导炎症反应的方式可能是激活中性粒细胞。在这一过程中,粘附分子CD44参与了中性粒细胞的粘附,其中TNF-α的表达依赖中性粒细胞的粘附分子CD44的表达,提示细胞因子TNF-α与粘附分子CD44之间存在细胞间信号转导。3、TNF-α和CD44在OME大鼠模型鼓室积液发生、发展中起重要协同作用。
[Abstract]:Objective : OME media with effusion ( OME ) is a chronic non - suppurative inflammation characterized by effusion of tympanic cavity . The pathogenesis is not clear . It is generally believed to be related to mechanical obstruction , infection and immune response in SD rats . Methods : Forty - four adult healthy SD rats were established , and three experimental groups were established in normal control group , control group ( PBS ) group and OME model group .
In the control group ( PBS ) , 16 SD rats were injected with phosphate buffer ( PBS ) in the middle ear cavity , and SD rats were sacrificed at 4 days , 4 days , 7 days and 15 days .
After lipopolysaccharide ( LPS ) injection in the middle ear cavity , the OME model group corresponds to the operation control ( PBS ) group , and the auditory bubble mucosa tissues and the rat auditory vesicle washing liquid line detection are taken at four different time points ( each time period is more selected into one SD rat , so that the number of experimental animals caused by animal death in the molding process is not sufficient ) . The levels of TNF - 伪 in the normal control group were measured by means of immunohistochemical SP method . Results : 1 . The concentration of TNF - 伪 in the normal control group was 148.858ng / ml , 239.898ng / ml , 135.753ng / ml , 101.830 ng / ml . The concentration of TNF - 伪 in the experimental group was significantly higher than that in the control group .
There were no obvious changes in the cells of the auditory cells in the two days , 4 days , 7 days and 15 days after the operation control ( PBS ) group , and only a small amount of inflammatory cell infiltration was found in the mucosa .
The expression of TNF - 伪 and CD44 in normal control group was significantly correlated with the expression of TNF - 伪 and CD44 in normal control group .

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.21

【引证文献】