糖皮質(zhì)激素對分泌性中耳炎模型水通道蛋白1表達(dá)的調(diào)控
發(fā)布時(shí)間：2019-04-03 12:55
【摘要】：目的 1、探索單純咽鼓管阻塞建立分泌性中耳炎動物模型的方法，為分泌性中耳炎急性期的相關(guān)實(shí)驗(yàn)研究提供一種理想的動物模型。 2、探尋水通道蛋白1在正常和分泌性中耳炎豚鼠中耳腔的表達(dá)和定位，以及在分泌性中耳炎病程中的表達(dá)變化，同時(shí)在蛋白水平觀察糖皮質(zhì)激素對水通道蛋白1表達(dá)的調(diào)控作用。方法 1、健康雄性豚鼠22只，左側(cè)為實(shí)驗(yàn)耳，右側(cè)為對照耳，經(jīng)軟腭切開膨脹海綿阻塞左側(cè)咽鼓管咽口建立可逆性分泌性中耳炎動物模型，耳內(nèi)鏡下觀察記錄鼓膜形態(tài)和鼓室積液情況，分別于術(shù)后第7天、第14天及第21天各獲取6只造模成功的豚鼠聽泡，探查術(shù)后膨脹海綿留置情況，HE染色觀察急性分泌性中耳炎病程中中耳黏膜的病理改變。 2、用免疫組織化學(xué)的方法觀察正常和分泌性中耳炎豚鼠中耳腔水通道蛋白1的表達(dá)和分布情況。 3、造模成功的豚鼠20只，隨機(jī)分為Dexa組（治療組）和OME組（非治療組），，每組10只。Dexa組術(shù)后第7天起，每天腹腔注射地塞米松5mg/kg，持續(xù)7天。術(shù)后第14天處死2組采集標(biāo)本。同時(shí)取5只正常豚鼠的雙側(cè)聽泡做為NC組（正常對照）。用免疫印跡方法在蛋白水平分別檢測3組水通道蛋白1表達(dá)量且比較有無差異。結(jié)果 1、造模組22耳中20耳分別于術(shù)后第3～7天出現(xiàn)鼓室漿液性滲出，造模成功率為90.9%，病理改變表現(xiàn)為鼓室黏膜增厚，黏膜下血管擴(kuò)張，淋巴細(xì)胞浸潤，近咽鼓管鼓室口部纖毛低矮，數(shù)量減少甚至脫落。術(shù)后14 d膨脹海綿已基本排出，14～18 d中耳積液消失，同時(shí)組織形態(tài)逐漸恢復(fù)正常。病程中對側(cè)對照耳鼓室無積液，亦無明顯病理改變。 2、水通道蛋白1在正常和分泌性中耳炎豚鼠中耳腔黏膜內(nèi)有明確表達(dá)，中耳腔黏膜下層的毛細(xì)血管內(nèi)皮和成纖維細(xì)胞均有水通道蛋白1分布。分泌性中耳炎豚鼠中耳腔的表達(dá)更為顯著。 3、3組的豚鼠中耳腔黏膜中，抗水通道蛋白1抗體在1：3000的工作濃度下識別出28kDa條帶。獨(dú)立樣本t檢驗(yàn)兩兩比較提示3組的蛋白表達(dá)量皆有差異。NC組的蛋白表達(dá)水平高于另外2組，Dexa組的蛋白表達(dá)水平顯著高于OME組(t=2.733，P0.05)。 結(jié)論 1、經(jīng)軟腭切開膨脹海綿咽鼓管阻塞法可成功建立豚鼠急性分泌性中耳炎模型，該分泌性中耳炎可于膨脹海綿自行排出后2周逐漸自愈。 2、水通道蛋白1在豚鼠中耳腔黏膜有表達(dá)，參與中耳腔的液體平衡過程。 3、水通道蛋白1在分泌性中耳炎自然轉(zhuǎn)歸過程中表達(dá)水平發(fā)生變化。糖皮質(zhì)激素可上調(diào)水通道蛋白1的表達(dá)，并且可能是通過此機(jī)制協(xié)同實(shí)現(xiàn)對分泌性中耳炎治療作用。
[Abstract]:Aim 1. To explore the method of establishing the animal model of secretory otitis media by simple eustachian tube obstruction, and to provide an ideal animal model for the experimental study of secretory otitis media in the acute stage. 2. To explore the expression and localization of aquaporin-1 in the middle ear cavity of guinea pigs with normal and secretory otitis media, and the changes of expression of aquaporin-1 in the course of secretory otitis media. The effects of glucocorticoid on the expression of aquaporin 1 were also observed at the protein level. Methods 1. Twenty-two healthy male guinea pigs with experimental ears on the left and control ears on the right were used to establish reversible secretory otitis media model through soft palate incision and swelled sponge blocking the pharynx mouth of the left pharynx tube, and the animal model of reversible secretory otitis media was established. The morphology of tympanic membrane and effusion of tympanum were observed and recorded under endoscope. Six guinea pig auditory vesicles were obtained on the 7th day, 14th day and 21st day after operation, respectively, and the indwelling condition of swelling sponge after operation was explored. HE staining was used to observe the pathological changes of ear mucosa in the course of acute secretory otitis media. 2. The expression and distribution of aquaporin 1 in the middle ear of guinea pigs with normal and secretory otitis media were observed by immunohistochemical method. 3. 20 guinea pigs were randomly divided into two groups: Dexa group (treatment group) and OME group (non-treatment group), 10 guinea pigs in each group. Dexa group received intraperitoneal injection of dexamethasone 5 mg / kg daily for 7 days from the 7th day after operation, and 10 guinea pigs in each group received intraperitoneal injection of dexamethasone (5 mg / kg) per day for 7 days. Two groups were sacrificed on the 14th day after operation. At the same time, the bilateral auditory vesicles of 5 normal guinea pigs were taken as NC group (normal control). The expression of aquaporin-1 in the three groups was detected by Western blot at the protein level and there was no difference in the expression of aquaporin-1. Results 1 in 20 ears of 22 ears in the model group, tympanic serous exudation occurred on the 3rd to 7th day after operation, and the success rate of the model was 90.9%. The pathological changes were as follows: thickening of tympanic mucosa, dilation of submucosal vessels and infiltration of lymphocytes. The cilia of the tympanic orifice near the eustachian tube was low and the number decreased or even fell off. The swelling sponge was basically discharged on the 14th day after operation, the middle ear effusion disappeared on the 14th day after operation, and the histologic morphology gradually returned to normal at the same time. There was no effusion in the contralateral tympanic chamber and no obvious pathological changes in the course of the disease. 2. Aquaporin-1 was clearly expressed in the middle ear mucosa of normal and secretory otitis media guinea pigs, and aquaporin-1 distribution was found in both capillary endothelium and fibroblasts of the submucosa of the middle ear cavity. The expression of secretory otitis media in the middle ear cavity of guinea pigs was more significant. 3. Anti-aquaporin-1 antibody detected 28kDa bands in the middle ear mucosa of guinea pigs in 3 groups at the working concentration of 1? 3 000. The independent sample t-test showed that the protein expression level in NC group was higher than that in the other two groups, and the protein expression level in Dexa group was significantly higher than that in OME group (t = 2.773, P0.05). The expression level of protein in Dexa group was significantly higher than that in OME group (t = 2.733, P0.05). Conclusion 1. Acute secretory otitis media model of guinea pigs can be successfully established by soft palate incision and swelling sponge eustachian tubule obstruction. The secretory otitis media can heal itself 2 weeks after expandable sponge. 2 weeks after the swelling sponge is discharged by itself, the model of secretory otitis media can be established successfully. 2. Aquaporin-1 was expressed in the middle ear mucosa of guinea pigs and was involved in the process of fluid balance in the middle ear cavity of guinea pigs. 3. The expression level of aquaporin-1 in secretory otitis media was changed during the course of spontaneous prognosis. Glucocorticoid can up-regulate the expression of aquaporin-1, and may be a synergistic mechanism for the treatment of secretory otitis media.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R764
本文編號：2453231
[Abstract]:Aim 1. To explore the method of establishing the animal model of secretory otitis media by simple eustachian tube obstruction, and to provide an ideal animal model for the experimental study of secretory otitis media in the acute stage. 2. To explore the expression and localization of aquaporin-1 in the middle ear cavity of guinea pigs with normal and secretory otitis media, and the changes of expression of aquaporin-1 in the course of secretory otitis media. The effects of glucocorticoid on the expression of aquaporin 1 were also observed at the protein level. Methods 1. Twenty-two healthy male guinea pigs with experimental ears on the left and control ears on the right were used to establish reversible secretory otitis media model through soft palate incision and swelled sponge blocking the pharynx mouth of the left pharynx tube, and the animal model of reversible secretory otitis media was established. The morphology of tympanic membrane and effusion of tympanum were observed and recorded under endoscope. Six guinea pig auditory vesicles were obtained on the 7th day, 14th day and 21st day after operation, respectively, and the indwelling condition of swelling sponge after operation was explored. HE staining was used to observe the pathological changes of ear mucosa in the course of acute secretory otitis media. 2. The expression and distribution of aquaporin 1 in the middle ear of guinea pigs with normal and secretory otitis media were observed by immunohistochemical method. 3. 20 guinea pigs were randomly divided into two groups: Dexa group (treatment group) and OME group (non-treatment group), 10 guinea pigs in each group. Dexa group received intraperitoneal injection of dexamethasone 5 mg / kg daily for 7 days from the 7th day after operation, and 10 guinea pigs in each group received intraperitoneal injection of dexamethasone (5 mg / kg) per day for 7 days. Two groups were sacrificed on the 14th day after operation. At the same time, the bilateral auditory vesicles of 5 normal guinea pigs were taken as NC group (normal control). The expression of aquaporin-1 in the three groups was detected by Western blot at the protein level and there was no difference in the expression of aquaporin-1. Results 1 in 20 ears of 22 ears in the model group, tympanic serous exudation occurred on the 3rd to 7th day after operation, and the success rate of the model was 90.9%. The pathological changes were as follows: thickening of tympanic mucosa, dilation of submucosal vessels and infiltration of lymphocytes. The cilia of the tympanic orifice near the eustachian tube was low and the number decreased or even fell off. The swelling sponge was basically discharged on the 14th day after operation, the middle ear effusion disappeared on the 14th day after operation, and the histologic morphology gradually returned to normal at the same time. There was no effusion in the contralateral tympanic chamber and no obvious pathological changes in the course of the disease. 2. Aquaporin-1 was clearly expressed in the middle ear mucosa of normal and secretory otitis media guinea pigs, and aquaporin-1 distribution was found in both capillary endothelium and fibroblasts of the submucosa of the middle ear cavity. The expression of secretory otitis media in the middle ear cavity of guinea pigs was more significant. 3. Anti-aquaporin-1 antibody detected 28kDa bands in the middle ear mucosa of guinea pigs in 3 groups at the working concentration of 1? 3 000. The independent sample t-test showed that the protein expression level in NC group was higher than that in the other two groups, and the protein expression level in Dexa group was significantly higher than that in OME group (t = 2.773, P0.05). The expression level of protein in Dexa group was significantly higher than that in OME group (t = 2.733, P0.05). Conclusion 1. Acute secretory otitis media model of guinea pigs can be successfully established by soft palate incision and swelling sponge eustachian tubule obstruction. The secretory otitis media can heal itself 2 weeks after expandable sponge. 2 weeks after the swelling sponge is discharged by itself, the model of secretory otitis media can be established successfully. 2. Aquaporin-1 was expressed in the middle ear mucosa of guinea pigs and was involved in the process of fluid balance in the middle ear cavity of guinea pigs. 3. The expression level of aquaporin-1 in secretory otitis media was changed during the course of spontaneous prognosis. Glucocorticoid can up-regulate the expression of aquaporin-1, and may be a synergistic mechanism for the treatment of secretory otitis media.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R764
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