RAGE信号途径与老年性聋发病过程的关系研究

发布时间：2018-05-30 05:59

本文选题：老年性聋 + 晚期糖基化终产物受体　；参考：《福建医科大学》2012年硕士论文

【摘要】：老年性聋病因及发病机制复杂，主要是衰老、凋亡、氧化应激和线粒体突变四大假说，其中衰老的自由基学说被较多人所接受。但产生自由基的原因甚多，分子机制尚未统一，因此目前临床老年性聋的治疗不容乐观。RAGE与AGEs结合可引起细胞内氧化应激和转录因子NF-κB的激活，并可进一步增强RAGE表达[15]，从而引起持续的细胞损伤和功能紊乱。RAGE介导的氧化应激在中枢神经系统损伤性疾病中至关重要，而在衰老的内耳中研究较少。本研究拟通过老年性聋动物模型，探讨RAGE及其介导信号途径与老年性聋发生发展的关系。主要内容分以下三部分进行。第一部分：C57BL/6J小鼠年龄相关性听功能和内耳组织学的研究目的：研究不同月龄的C57BL/6J小鼠的听功能和内耳组织学特点，探讨老年性聋发病的动物模型。方法：将C57BL/6J小鼠分为2月龄（幼年）、4月龄（中）、10月龄（老年）组，每组10只，分别行ABR阈值检测听力水平，组织学切片观察耳蜗形态结构，基底膜铺片观察耳蜗Corti器。结果：（1）听力学检查示：C57BL/6J小鼠ABR反应阈值在2月龄组为10±1.24dBnHL；4月龄组时ABR反应阈值上升到18±1.87dBnHL，与2月龄组相比较，p0.05，有统计学意义；10月龄组听力损失60±2.91dBnHL，，与4月龄组相比，p0.01，差异有显著的统计学意义；(2) HE染色见螺旋神经节细胞随年龄增大逐渐减少，到10月龄时明显较2、4月龄减少，内外毛细胞不同程度丢失，血管纹在老年组的小鼠内耳中变细窄；（3）基底膜铺片示随年龄增长内外毛细胞由低回逐渐向顶回有所缺失，老年鼠外毛细胞缺失先于内毛细胞；结论：C57BL/6J小鼠随年龄增大呈老年性聋的听力变化。第二部分：C57BL/6J小鼠血清AGEs、内耳S100B和RAGE表达的相关性研究目的：探讨C57BL/6J小鼠内耳RAGE表达和AGEs、S100B是否具有相关性。方法：将C57BL/6J小鼠分为2月龄、4月龄、10月龄组，每组10只，采用ELISA法检测血清中AGEs含量，半定量RT-PCR检测S100B在耳蜗中的表达，Real time PCR检测RAGE在耳蜗中表达情况。结果：（1）2月龄组小鼠血清AGEs浓度是71.33±7.09ng/L，4月龄组105.44±12.1ng/L，10月龄组226.67±7.63ng/L，经两两比较，p0.05，差异有统计学意义；（2）S100B在2月龄小鼠耳蜗中的灰度值是31836±363,24.72,在10月龄小鼠耳蜗中的灰度值是67449.66±1988.61，P0.05，差异有统计学意义。（3）Real time PCR检测RAGEmRNA表达结果示：将2月龄组小鼠内耳RAGE表达量为1定为标准对照组，4月龄组小鼠内耳RAGE相对量1.61±0.07，10月龄组RAGE相对量3.13±0.08，两两比较p0.05，P均有统计学意义。结论：C57BL/6J小鼠随年龄增大血清AGEs含量增多，内耳S100B表达增高，而促使它们的受体RAGE表达增多，说明内耳RAGE表达和配体含量具有相关性。第三部分：RAGE介导的信号通路在老年性聋的作用研究目的：通过研究RAGE、NF-κB、P21在小鼠耳蜗中的表达，探讨RAGE信号途径与老年性聋发生的关系。方法：将C57BL/6J小鼠分为2月龄、4月龄、10月龄组，每组10只，免疫组化检测RAGE、NF-κB、P21在耳蜗中的定位表达，采用图像分析软件计算所染螺旋神经节细胞和血管纹的平均光密度值。结果：免疫组化结果见RAGE、NF-κB、P21主要在小鼠耳蜗的螺旋神经节细胞、Corti器和血管纹中表达；在耳蜗螺旋神经节细胞和血管纹中10月龄组平均光密度值比4月龄组大，4月龄组平均光密度值比2月龄大，p0.05，差异有统计学意义。结论：RAGE、NF-κB、P21在老化型小鼠耳蜗中均有表达且随年龄增大表达增多，说明RAGE、NF-κB、P21均参与老年性聋的发病过程，可能RAGE介导NF-κB、P21等相关蛋白导致听觉细胞丢失或损伤，表明RAGE介导的信号途径与老年性聋的发病过程有相关性。
[Abstract]:The etiology and pathogenesis of senile deafness are complex, mainly four hypotheses of aging, apoptosis, oxidative stress and mitochondrial mutation, of which the free radical theory of aging is accepted by many people. However, there are many reasons for the generation of free radicals and the molecular mechanism is not unified. Therefore, the treatment of clinical senile deafness can not be optimistic about the combination of.RAGE and AGEs. The activation of intracellular oxidative stress and transcription factor NF- kappa B can further enhance the RAGE expression of [15], thus causing persistent cell damage and dysfunction of.RAGE mediated oxidative stress in the central nervous system injury diseases, but less in the aging inner ear. This study is to explore RA through the senile deafness animal model. The relationship between GE and its mediating signal pathway and the occurrence and development of senile deafness is divided into three parts.
The first part: the study of age related auditory function and inner ear histology in C57BL/6J mice: To study the auditory and inner ear histology of C57BL/6J mice of different months of age and to explore the animal models of the onset of senile deafness.
Methods: the C57BL/6J mice were divided into 2 month old (young), 4 month old (middle), 10 month old (old) group and 10 in each group. The hearing level was measured by ABR threshold, and the cochlear morphology was observed by histological section. The cochlear Corti was observed by the basement membrane sheet.
Results: (1) the audiological examination showed that the threshold of ABR reaction in C57BL/6J mice was 10 + 1.24dBnHL, and the threshold of ABR reaction increased to 18 + 1.87dBnHL in the 4 month old group. Compared with the 2 month old group, P0.05 was statistically significant; the 10 month old group of hearing loss was 60 + 2.91dBnHL, and the difference was statistically significant compared with the 4 month old group, and (2) H. E staining showed that the spiral ganglion cells decreased gradually with age, at 10 month old, obviously less than 2,4 months, the internal and external hair cells were lost in different degrees, and the vascular lines were narrowed in the inner ear of the old mice. (3) the basal membrane sheets showed the loss of the inner and outer hair cells from the low back to the top, and the loss of outer hair cells in the aged rats. Prior to internal hair cells;
Conclusion: C57BL/6J mice showed hearing loss with age.
The second part: the correlation of C57BL/6J mice serum AGEs, S100B and RAGE expression in the inner ear: To investigate the expression of RAGE in the inner ear of C57BL/6J mice and the correlation between AGEs and S100B. Methods: C57BL/6J mice were divided into 2 month old, 4 month old, 10 month old, and 10 groups. The AGEs content in the serum was detected by ELISA method, and the semi quantitative RT-PCR was detected. The expression of B in the cochlea and Real time PCR were used to detect the expression of RAGE in the cochlea.
Results: (1) the concentration of AGEs in the 2 month old groups of mice was 71.33 + 7.09ng/L, 4 month old 105.44 + 12.1ng/L, 10 month old groups 226.67 + 7.63ng/L, and the difference was statistically significant after 22. (2) the gray value of S100B in the cochlea of 2 month old mice was 2 month old 363,24.72, and the gray value in the cochlea of 10 month old mice was 67449.66 + 1988.61, P0.05, The difference was statistically significant. (3) Real time PCR detection RAGEmRNA expression results showed that the 2 month old groups of mice internal ear RAGE expression of 1 was set as the standard control group, 4 month old groups of mice inner ear RAGE relative quantity 1.61 + 0.07,10 month group RAGE relative quantity 3.13 + 0.08, 22 P0.05, P all statistical significance. Conclusion: C57BL/6J mice with age increases. The serum AGEs level increased, the expression of S100B increased in the inner ear, and the expression of RAGE increased, indicating that the expression of RAGE in the inner ear was related to the ligand content.
The third part: the role of RAGE mediated signal transduction pathway in senile deafness.
Objective: To study the expression of RAGE, NF- kappa B and P21 in mouse cochlea, and to explore the relationship between RAGE signal pathway and the occurrence of senile deafness.
Methods: C57BL/6J mice were divided into 2 month old, 4 month old, 10 month old groups, each group of 10. Immunohistochemistry was used to detect RAGE, NF- kappa B, P21 in the cochlea, and the mean optical density of the infected spiral ganglion cells and vascular lines was calculated by the image analysis software.
Results: the results of immunohistochemistry were RAGE, NF- kappa B, P21 mainly in the spiral ganglion cells of the cochlea, Corti and vascular lines, and the average optical density of 10 month old groups in the cochlear spiral ganglion cells and vascular lines was larger than that of the 4 month old groups, and the average optical density of the 4 month old groups was larger than that of 2 month old, and the difference was statistically significant.
Conclusion: RAGE, NF- kappa B and P21 are expressed in the cochlea of aging mice and increase with age, indicating that RAGE, NF- kappa B, P21 are all involved in the pathogenesis of senile deafness, and RAGE mediates NF- kappa B, P21 and other related proteins lead to the loss or damage of auditory cells, indicating that the signaling pathway is related to the process of senile deafness. Sex.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764.4

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