【作者】 韩宇；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 【背景】随着工业化进程的加快,感音神经性耳聋已逐渐成为影响公众健康的重要社会问题之一。过度的噪音暴露可导致内耳机械性和代谢性损伤,严重影响了人类的生活质量,并且最终可导致噪声诱导的听力降低甚至缺失。然而,哺乳动物耳蜗毛细胞为特化终末细胞,损失后其再生能力十分有限,这也是导致永久性听力损失的原因。因此,发现新的哺乳动物内耳调控基因,阻止诱导毛细胞凋亡的信号分子很可能为感音神经性耳聋的治疗带来曙光。原癌基因c-myc是快速早期反应基因,其编码的细胞核蛋白调控细胞周期由G1期进入S期。通常认为c-myc基因在许多生物过程中发挥了重要作用,包括胚胎发育、细胞增殖、分化及凋亡等。此外,据报道静止细胞重返细胞周期过程中,c-myc基因起到募集转录因子的重要功能。该基因虽然如此重要,但在哺乳动物耳蜗中的研究却未见报道。因此,阐明c-myc基因在耳蜗中的作用为哺乳动物耳蜗毛细胞再生提供新的思路。【目的】探讨c-myc基因在噪声条件下对豚鼠听功能及耳蜗毛细胞形态的作用及其可能的分子机制。【方法】构建含c-myc基因的腺病毒表达载体(Ad.c-myc-EGFP),并通过耳蜗冰冻切片观察报告基因表达和Western blot方法鉴定转染后c-Myc表达量的变化。于噪声暴露前4天通过耳蜗显微注射术将Ad.c-myc-EGFP注射入豚鼠耳蜗鼓阶内,并以空白腺病毒载体(Ad. EGFP)做对照。将各组豚鼠暴露于110 dB SPL的白噪声,频谱分析能量集中在0.25-6.4kHz,主要集中在4 kHz左右,8 h/day,连续7天。分别在噪声暴露前及噪声暴露后的第7天通过听性脑干反应(ABR)检测豚鼠听功能的情况,并利用基底膜铺片和扫描电镜等方法观察耳蜗毛细胞形态及超微结构的变化。通过基因芯片和生物信息学分析进一步探讨噪声对哺乳动物内耳基因表达谱的影响,并利用实时定量PCR验证基因芯片的结果;通过Tunel染色评估凋亡在噪声性损伤中的作用;利用免疫组化方法观察筛选到的目标基因在耳蜗中的表达与定位,从而推测c-myc基因在噪声条件下可能发挥作用的分子机制。【结果】成功构建了Ad.c-myc-EGFP腺病毒载体,并证明它可以有效干预耳蜗内c-Myc蛋白的表达水平。通过体内实验观察了c-myc基因对耳蜗的作用,主要表现为:相同噪声暴露后,Ad.c-myc-EGFP注射组听阈提高显著低于Ad.EGFP组;Ad.c-myc-EGFP注射组外毛细胞形态改变及纤毛缺失率明显低于Ad.EGFP注射组;扫描电镜观察亦表明Ad.c-myc-EGFP注射组外毛细胞超微结构损伤程度明显小于Ad.EGFP对照组。本实验进一步利用基因芯片探讨噪声所致内耳分子的变化:噪声可以导致539个基因发生变化,其中279个基因上调,260个基因下调。通过生物信息学分析:这些差异基因主要涉及免疫系统、应激、生物代谢、节律调节及凋亡等相关基因。实时定量PCR鉴定了差异基因倍数大于5倍以上以及与免疫调节相关的基因,结果表明9种基因与基因芯片的趋势相一致,上调的有Reg3b,Lcn2,Nob1,Serpina3n,Hamp,Lbp,C3;下调的为Myh4,F2。Tunel染色观察表明:噪声可以诱导耳蜗内、外毛细胞及螺旋神经节细胞发生凋亡。因此,我们推测c-myc基因在噪声条件下对豚鼠听力及耳蜗毛细胞损伤的推迟作用很可能是由于c-myc基因上调了保护性基因而下调了诱导耳蜗细胞凋亡基因的表达而发挥作用,但c-myc基因与这9种分子在耳蜗细胞内的具体调节方式及作用关系还有待进一步阐明。此外,我们进一步利用免疫组化观察到Nob1基因编码蛋白在噪声暴露后大鼠耳蜗内呈阳性表达,阳性细胞主要集中在螺旋神经节和内、外毛细胞;血管纹细胞也有少量表达;而噪声暴露前耳蜗内未见Nob1蛋白的明显表达。因此,推测Nob1基因作为转录因子可能参与耳蜗内基因的调控,是一个新的内耳调控基因,其作用及功能有待深入研究。【结论】c-myc基因减弱了噪声诱导的豚鼠听功能损伤及毛细胞形态的改变,并可能通过调节Reg3b,Lcn2,Nob1,Serpina3n,Hamp,Lbp,C3,Myh4,F2基因及凋亡相关分子发挥作用。更多还原

【Abstract】 【Background】With industrialization of our society, acoustic trauma is becoming an important public health problem. Excessive noise exposure inflicts both mechanical and metabolic damage to the inner ear, which has a significant impact on the quality of life and may lead to noise induced hearing loss (NIHL). In humans, loss of cochlear hair cells is permanent, because the auditory cells are considered as terminal cells, without the ability of regeneration. One strategy for preventing NIHL might be to develop therapeutics that block the cellular signaling leading to cell death following trauma. Therefore, manipulating related molecules in mammalian cochleae could provide a new avenue to restore hearing.The proto-oncogene c-myc, an immediate-early gene, encodes a nuclear protein that is known to be a major regulator for the G1 to S transition. It is genenrally accepted that this gene has a major role in multiple processes, including embryonic development, cell proliferation, differentiation, and apoptosis. In addition, it was reported that c-myc might recruit transcription factors when quiescent cells were stimulated to reenter the cell cycle. However, the funtion of c-myc gene in the mammalian cochlea is still unclear. Therefore, it may provide insights into the regeneration of hair cells in mammalian. It is necessary to clarify the function of this gene in the cochlea.【Aims】The study was designed to investigate the function and mechanisms of c-myc gene under noise condition, from the aspect of cochlear morphological changes and auditory function.【Methods】We had established the adenoviral vector of c-myc gene (Ad.c-myc-EGFP), and western blot and frozen section were used to detecte the expression of c-Myc in inner ear. Four days before noise exposure, the adenovirus suspension was delivered into the scala tympani of guinea pigs. The empty adenoviral vector (Ad. EGFP) was injected as control. Then, all subjects were exposed to 110 dB SPL white noise, mainly concentrated on about 4-kHz frequency, for 8 h/day, 7 days consecutively. Auditory thresholds were assessed by auditory brainstem response (ABR), prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. Then, microarray and bioinformatics were used to investigate the mechanisms underlying this gene’s role on noise induced hearing loss, and real-time PCR was used to identify the results of genechip. Tunel staining was used to clarify the role of apoptosis in NIHL. The expression and location of significant genes in the cochlea were examined by immunohistochemistry. Thus, we speculated the molecular mechanism of c-myc gene in the cochlea under noisy condition.【Results】We had successfully established the adenoviral vector of c-myc gene, and it could efficiently upregulate the expression of c-Myc protein. The roles of c-myc gene in the cochlea indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Aslo, the ultrastructure changes were severe in the Ad. EGFP group, but were not obvious in the Ad.c-myc-EGFP group. As the results of microarray showed, noise might mediate 539 kinds of genes, including 279 kinds of genes significantly upregulated and 260 kinds of genes significantly downregulated. These genes mainly involved in immune system, stress, homeostatic process, rhythm and apoptosis. As identified by real time PCR, noise may upregulate Reg3b,Lcn2,Nob1,Serpina3n,Hamp,Lbp,C3 genes, and downregulated Myh4,F2 genes. The result of tunel staining indicated noise could induce apoptosis of IHCs, OHCs and SGCs. Therefore, we speculated the roles of c-myc gene in the guinea pig’s cochlea maybe due to upregulate protective genes or downregulate apoptosis related genes. However, the definite mechnisims had yet to be further clarified. Especially, NOB1 expression was detected mainly in spiral ganglion cells (SGCs), inner hair cells (IHCs) and outer hair cells (OHCs) after noise exposure, but the expression of NOB1 was not detected in normal ones. We speculated that nob1 gene as a transcription factor is likely involved in the regulation of genes within the cochlea, and it is a new regulatory gene of inner ear, its roles need further investigation. 【Conclusions】c-Myc might play an unexpected protective roles in hearing functional and morphological protection from acoustic trauma, through regulation of Reg3b,Lcn2,Nob1,Serpina3n,Hamp,Lbp,C3,Myh4,F2 and apoptosis related genes.更多还原