【作者】 汪芹；

【导师】 伍伟景；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 研究背景：跨膜丝氨酸蛋白酶3(TMPRSS3)为Ⅱ型跨膜丝氨酸蛋白酶家族成员,是第一个被发现的致聋的蛋白酶,其基因缺陷是非综合征型常染色体隐性遗传性聋DFNB8/10的遗传学基础,至今已发现该基因14种突变致聋形式,但对于该蛋白酶的功能及其作用机制仍不清楚。目的：本研究在增龄相关性听力损失和卡那霉素致聋的SD大鼠模型上,通过检测耳蜗中TMPRSS3基因的表达特点,初步探讨其在增龄相关性听力损失及药物中毒性聋中的作用。方法：用于卡那霉素致聋研究的大鼠均为3月龄,随机分为四组：Ⅰ组(对照组)：注射生理盐水14天；Ⅱ组(卡那霉素3日组)：肌注硫酸卡那霉素,连续3天；Ⅲ组(卡那霉素7日组)：肌注硫酸卡那霉素,连续7天；Ⅳ组(卡那霉素14日组)：肌注硫酸卡那霉素,连续14天。用于增龄相关性聋的大鼠按月龄分为三组：3月龄组、12月龄组、24月龄组。通过听性脑干反应(ABR)测试确定各组8 kHz和16kHz的听反应阈。应用免疫组化技术检测各组大鼠耳蜗中TMPRSS3蛋白的表达与分布情况。应用Western blot技术检测耳蜗中TMPRSS3蛋白表达水平。应用荧光定量逆转录多聚酶链反应(RT-PCR)对大鼠耳蜗中TMPRSS3基因的mRNA表达水平进行检测。结果：1.卡那霉素致聋大鼠：(1)听功能改变：ABR反应阈由高到低依次为：卡那霉素14日组>卡那霉素7日组>卡那霉素3日组,而对照组与注射3日组之间ABR反应阈值无明显差异。(2)免疫组化染色：TMPRSS3蛋白在各组SD大鼠耳蜗中均有表达,随注射时间延长,染色阳性程度变弱。(3)Western blot检测：TMPRSS3蛋白在耳蜗的表达浓度随着注射时间延长而减弱。(4)RT-PCR分析：抽提耳蜗组织总RNA电泳结果OD260/280在1.9-2.0之间。TMPRSS3基因的mRNA表达水平随注射时间延长而逐渐下降。2.增龄相关性听力损失大鼠：(1)听功能改变：在16kHz, ABR反应阈由高到低依次为：24月龄组>12月龄组>3月龄组,各组间均有显著差异(P<0.01)。在8kHz,24月龄组较3月龄及12月龄组大鼠阈值增高,而3月龄和12月龄组之间无显著差异；(2)免疫组化染色：随月龄增长,TMPRSS3蛋白耳蜗染色阳性程度变弱。(3)Western blot检测：大鼠耳蜗中TMPRSS3蛋白.表达浓度随年龄增长而减弱。(4)RT-PCR分析：TMPRSS3 mRNA表达水平随月龄增长而减弱,各组间均有显著差异。结论：TMPRSS3在SD大鼠耳蜗螺旋器和螺旋神经节细胞均有表达。SD大鼠耳蜗中TMPRSS3蛋白和mRNA表达水平随注射硫酸卡那霉素时间增长和月龄增长而逐渐降低。各实验组TMPRSS3蛋白和mRNA表达水平与听功能保存程度基本一致。上述结果提示TMPRSS3可能在药物中毒和老年性聋的发生中起作用。研究背景：表皮钠通道蛋白(ENaC)属于ENaC/Deg家族,是由3个同源亚基α、β、γ组成的多聚体,分布于肾小管、结肠、呼吸道上皮细胞表面及耳蜗等处,在耳蜗其主要作用是维持内淋巴的低钠浓度。2002年Guipponi等利用体外爪蟾卵表达系统,证实了TMPRSS3能催化激活ENaC。也有研究发现TMPRSS3致聋的突变体无法激活ENaC。但目前对于ENaC在调节内耳内淋巴代谢、维持内耳正常功能方面的机制尚不清楚。目的：本研究在对增龄相关性听力损失和卡那霉素致聋的SD大鼠,通过检测耳蜗中ENaC-α基因的表达特点,初步探讨其在老年性聋及药物性听力损失中的作用,及与TMPRSS3的关系。方法：动物分组处理同第一部分,应用免疫组化技术,检测各组SD大鼠耳蜗中ENaC-α蛋白的表达与分布情况。应用Western blot技术,检测两类致聋模型大鼠耳蜗中ENaC-α的蛋白表达水平。应用荧光定量逆转录多聚酶链反应(RT-PCR),对大鼠耳蜗中ENaC-α基因的mRNA表达水平进行检测。结果：1.卡那霉素致聋大鼠：(1)免疫组化染色：ENaC-α蛋白在各组SD大鼠耳蜗中均有表达,注射14日组大鼠较注射3日、7日组及对照组的大鼠耳蜗染色明显减弱,但注射3日、7日组及对照组之间无显著差异；(2)Western blot检测：对照组及注射3日、7日的大鼠耳蜗蛋白表达浓度无明显改变,注射14日组较前三组ENaC-α蛋白浓度明显降低。(3)RT-PCR分析：抽提耳蜗组织总RNA电泳结果OD260/280在1.9-2.0之间。注射3日、7日及对照组的大鼠耳蜗mRNA表达水平无明显改变,注射14日组ENaC-αmRNA表达水平较前三组减弱,有统计学显著差异。2.增龄相关听力损失大鼠：(1)免疫组化染色：SD大鼠随月龄增长,耳蜗ENaC-α蛋白染色阳性程度减弱。(2)Western blot检测：ENaC-α蛋白表达浓度随大鼠月龄增长而降低。(3)RT-PCR分析：大鼠耳蜗ENaC-αmRNA表达水平随月龄增长逐渐减弱,有显著差异。结论：ENaC-α在SD大鼠耳蜗Corti’s器和螺旋神经节细胞均有表达。卡那霉素致聋大鼠耳蜗中ENaC-α蛋白和mRNA表达水平在给药后期明显降低；增龄相关听力损失大鼠随月龄增大而降低。ENaC-α的变化与TMPRSS3有相关性,提示TMPRSS3可能通过调节ENaC-α而起作用。更多还原

【Abstract】 Backgrounds TMPRSS3 (transmembrane protease serine 3) is a member of transmembraneⅡserine proteases(TTSPs). TMPRSS3 is the first-found protease that its mutation could lead to deafness and is mutated in non-syndromic autosomal recessive deafness(DFNB8/10).The 14 pathogenic changes are found to date. However, it is not clear about the function of TMPRSS3.Objective On the basis of researching rats with age-related and kanamycin-induced hearing loss, to investigate the expression of TMPRSS3 in the SD rats cochlea, and to explore the roles of TMPRSS3 in sensorineural deafness.Methods SD rats which were used for kanamycin-induced hearing loss were 3-month-old and were divided into four groups in this study. GroupⅠ(control), treated with normal saline for 14 days. GroupⅡ(KN3d), treated with kanamycin sulfate for 3 days. GroupⅢ(KN7d), treated with kanamycin sulfate for 7 days. GroupⅣ(KN14d), treated with kanamycin sulfate for 14 days. Rats used for age-related hearing loss were divided into three groups:3-、12- and 24- month-old. The auditory function was evaluated by evoked auditory brainstem responses (ABR). Paraffin-embedded immunohistochemistry was used for evaluation of TMPRSS3 expression in the cochlea. The protein was extracted from the cochlea tissues, and TMPRSS3 protein levels in the cochlea were detected by Western blot assay. The RNA was extracted from the cochlea tissues and TMPRSS3 mRNA levels in the cochlea were measured by reverse transcription-polymase chain reaction (RT-PCR).Results 1. Rats with kanamycin-induced hearing loss:(1) Auditory function:The order of the ABR thresholds from high to low was KN14d > KN7d> KN3d. (2) Immunohistochemistry:A positive reaction for TMPRSS3 staining was found in the cochlea of each group. The expression of TMPRSS3 immunoreactivity prominently decreased following the increase of injection days. (3) Western blot assay showed that the levels of TMPRSS3 in the cochlea of SD rats decreased with injection days. (4) RT-PCR assay:RT-PCR demonstrated that the mRNA expression of TMPRSS3 decreased with injection days.2. Rats with. age-related hearing loss:(1) Auditory function:ABR thresholds increased with aging at 16 kHz. There were significant differences between each two groups. At 8 kHz, ABR thresholds of 24-month-old rats were higher than 3-and 12-month-old rats, there wasn’t significant differences between 3-and 12-month-old rats. (2) Immunohistochemistry:The expression of TMPRSS3 immunoreactivity prominently decreased following age increase. (3) Western blot assay: TMPRSS3 protein in the cochlea of SD rats decreased with aging. (4) RT-PCR assay:RT-PCR demonstrated that the mRNA expression of TMPRSS3 decreased with aging.Conclusions Positive reaction for TMPRSS3 staining was found in the cochlea of normal SD rats, mainly observed in the spiral ganglion and organ of Corti. The protein and mRNA of TMPRSS3 decreased with kanamycin administration and aging, and were basically consistant with the auditory function. These results indicated that TMPRSS3 may take part in the processes of hearing loss. Backgrouds ENaC (epithelial sodium channel) is a member of ENaC/Deg, and hasα、β、γ.subunits. ENaC is expressed in the distal nephron、colon、lung、cochlea and so on. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. TMPRSS3 mutants failed to undergo proteolytic cleavage and activate ENaC. However, it is not clear about ENaC protein’s function in regulating the metabolism of endolymph and maintenance of normal hearing.Objective To investigate the expression of ENaC-αin the SD rats with age-related and kanamycin-induced hearing loss, and to explore the roles of ENaC-a in the two kinds of hearing loss, whether it has correlation with TMPRSS3.Methods Group dividing is as same as PartⅠ. Paraffin-embedded immunohistochemistry was used for evaluation of ENaC-a expression in the cochlea. The protein was extracted from the cochlea tissues, and ENaC-a protein levels in the cochlea were detected by Western blot assay. The RNA was extracted from the cochlea tissues and ENaC-a mRNA levels in the cochlea were measured by reverse transcription-polymase chain reaction (RT-PCR).Results 1. Rats with kanamycin-induced hearing loss: (1)Immunohistochemistry:A positive reaction for ENaC-a staining was found in the cochlea of each group. The expression of ENaC-a immunoreactivity prominently decreased in the KN14d group compared with KN6d、KN3d and control groups, but there wasn’t significant differences among KN6d、KN3d and control groups. (2)Western blot assay showed that the levels of ENaC-a protein decreased in the KN14d group compared with KN3d、KN7d and control groups, but there wasn’t significant differences among KN3d、KN7d and control groups. (3)RT-PCR assay:There wasn’t significant differences among KN3d、KN7d and control groups, ENaC-a decreased in the KN14d group compared with KN3d、KN7d and control groups.2. Rats with age-related hearing loss:(1)Immunohistochemistry:The expression of ENaC-a immunoreactivity prominently decreased following age increase. (2)Western blot assay showed that ENaC-a protein in the cochlea of SD rats decreased with aging. (3)RT-PCR assay: RT-PCR demonstrated that the mRNA expression of ENaC-a in the cochlea of SD rats decreased with aging.Conclusions Positive reaction for ENaC-αstaining was found in the cochlea of normal SD rats, mainly observed in the the spiral ganglion and organ of Corti. The protein and mRNA of ENaC-αdecreased with injection days and aging. These results has some correlation with TMPRSS3 and indicated that TMPRSS3 may regulate the expression of ENaC.更多还原