【作者】 刘火旺；

【导师】 孙虹；

【作者基本信息】 中南大学 ， 耳鼻咽喉头颈外科， 2010， 博士

【摘要】 目的：应用功能分类基因芯片技术研究复发性鼻息肉组织的相关基因,并探讨其在鼻息肉发病和复发中的作用。方法：分别抽提检测10例鼻息肉组织、10例复发性鼻息肉组织、10例慢性鼻窦炎组织和10例正常鼻黏膜组织的总RNA,合成cDNA,在标准96孔PCR反应仪中进行实时定量PCR实验,用扫描仪扫描芯片荧光信号,用2-△△Ct计算JAK-STAT信号通路、MAPK信号通路和NF-κB信号通路中实验组与对照组对应基因的表达差异。为了验证这些发现的可重复性,同时也为了检验我们建立的实验体系的可靠性,我们严格按照第一次实验的设计,重复进行了第二次实验。结果：两次实验共得出与正常对照组比较上调2倍以上的差异基因14个,其中JAK-STAT信号通路9个、MAPK信号通路2个,NF-κB信号通路3个。在差异表达的基因中,主要包括信号转录与转导激活因子、转录因子、细胞因子信号转导抑制因子、表皮生长因子相关因子、细胞周期调节因子、补体因子等。其中可能反映鼻息肉复发状态的基因7个；可能反映鼻息肉状态的基因4个；可能反映鼻黏膜炎症状态的基因1个。结论：用功能分类基因芯片筛选鼻息肉、复发性鼻息肉、慢性鼻窦炎和正常鼻黏膜组织间存在的差异基因是可行的,这将为寻找鼻息肉分类和预后评估的客观实验室指标提供新的方法。目的：检测细胞周期蛋白D1(Cyclin D1)和抑瘤素M(Oncostatin M, OSM)蛋白在复发性鼻息肉、鼻息肉、慢性鼻窦炎及正常中鼻甲黏膜组织中的表达程度的差异,探讨其在复发性鼻息肉发病机制中的作用。方法：采用蛋白质印迹法分别检测6例复发性鼻息肉、6例鼻息肉、6例慢性鼻窦炎及6例正常中鼻甲黏膜组织中Cyclin D1和OSM的蛋白表达；免疫组织化学技术检测6例复发性鼻息肉、6例鼻息肉、6例慢性鼻窦炎及6例正常中鼻甲黏膜组织中Cyclin D1蛋白的表达及定位,并比较表达程度的差异。结果：(1)免疫组化显示Cyclin D1蛋白在复发性鼻息肉、鼻息肉、慢性鼻窦炎及正常中鼻甲黏膜组织中均有表达,Cyclin D1蛋白主要位于鼻黏膜上皮细胞和腺上皮细胞的细胞核,黄色或棕黄色染色为阳性。蛋白质印迹显示Cyclin D1蛋白和OSM蛋白在复发性鼻息肉、鼻息肉及慢性鼻窦炎组织中的表达均高于正常中鼻甲黏膜组织。(2)复发性鼻息肉组织中Cyclin D1和OSM蛋白表达水平高于鼻息肉、慢性鼻窦炎及中鼻甲组织,差异有统计学意义(P值均<0.05)。结论：Cyclin D1和OSM在复发性鼻息肉组织中表达增高,提示它们可能在鼻息肉的复发中起着重要的作用,可作为检测鼻息肉复发的候选指标。更多还原

【Abstract】 Chapter 1The study of the biomarkers in JAK-STAT, NF-κB and MAPK signaling pathway of the recurrent nasal polyps tissues with functional gene chips techniqueObjective:To detect the differentially expressed genes in the signaling pathway of JAK-STAT, NF-κB and MAPK in the recurrent nasal polpys through functional gene chips technique and evaluate the significance of these detected genes in the pathogenesis and recurrence of nasal polyps.Methods:The cDNAs were synthesized from total RNA which were extracted from 4 groups (10 cases respectively) including nasal polyps, recurrent nasal polyps, chronic sinusitis and normal nasal mucosa subjects. The cDNA were then taken into standard 96-well PCR equipment for real time PCR. The fluorescence signals of gene chip were obtained through a scanner. The expression difference of compared genes between experimental and normal groups in singaling pathway of JAK-STAT,MAPK and NF-κB was calculated by 2-ΔΔCt. These experiment processes were repeated strictly accorded to the experiment design to assure the stability and reliability of the final data.Results:There were 14 differentially expressed genes which were up-regulated up to more than 2-fold compared with the normal group, including 9 genes in JAK-STAT signaling pathway,2 genes in MAPK signaling pathway and 3 genes in NF-κB signaling pathway. These differentially expressed genes were singal transduction and transcription activation factor, transcription factor, cytokine transduction inhibition factor, epidermal growth associated factor, cell cycle regulation factor and complement factor, etc. Among the 14 candidates, there are seven genes whose upregulation may specially reflects the recurrent status of the nasal polyps, four genes for the status of nasal polpys and one for the inflammation status of nasal mucosa. Conclusions:It is feasible to screening the differentially expressed genes among the nasal polyps, recurrent nasal polyp, chronic sinusitis and normal nasal mucosa by means of functional classification gene chip technique. It will provide a new method in development of the objective experimental indicator for the classification and prognosis evaluation of nasal polyps.Chapter 2The expression and significance of Cyclin D1 and Oncostatin M in the recurrent nasal polypsObjective:To detect the expressions of Cyclin D1 and Oncostatin M proteins in recurrent nasal polyps, nasal polyps, chronic sinusitis and normal nasal mucosa so as to study their roles in the pathogenesis of the recurrent nasal polyps.Methods:The protein expression of Cyclin D1 and Oncostatin M were detected by Western blotting in recurrent nasal polyps, nasal polyps, chronic sinusitis and normal nasal mucosa cases (6 cases for each group). The expression and localization of Cyclin D1 were immunohistochemi-cally observed in the above four groups.Results:(1) Cyclin D1 protein was expressed in all of the specimen of the four groups, and predominantly stained in the nuclei of both epithelial cells of nasal mucosa and the underlying gland cells. The positive staining was yellow or claybank. By western blotting the expressions of Cyclin D1 protein and OSM protein in the recurrent nasal polyps, nasal polyps and chronic sinusitis were higher than that in normal middle turbinate mucosa. (2) The protein expressions of OSM and Cyclin D1 in the recurrent nasal polyps were significantly higher than that in other groups (p<0.05).Conclusions:The expressions of Cyclin D1 and OSM were up-regulated in the tissue of recurrent nasal polyps, which indicate that they may play the important roles in the development of recurrent nasal polyps, and may be utilized as the candidate biomarkers for detecting the susceptibility of recurrent nasal polyps.更多还原