P19ink4d蛋白在耳聋发生和发展中的表达及研究
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摘要
目的:建立稳定有效的噪声性听觉损伤模型,并观察P19ink4d在小鼠耳蜗中的变化,为探索噪声性聋的发病机制提供新的思路。对极重度感音神经性聋患者进行P19ink4d基因测序,试图发现P19ink4d基因突变致聋的家系,建立国人P19ink4d基因突变谱。方法:将CBA/J小鼠在101dB的宽带噪声持续暴露2h后,通过小鼠听性脑干反应(ABR)阈值测量、耳蜗基膜铺片及DAB毛细胞染色方法检测毛细胞的病理损伤过程,并采用免疫荧光法和Western blot检测基膜P19ink4d蛋白的表达,检测噪声对耳蜗毛细胞形态、数量及对P19ink4d蛋白的影响。在400例重度或极重度感音神经性聋儿童患者中,采用遗传性聋基因芯片排除由4个常见致聋基因突变致聋的患者,对未携带或仅带有单个杂合突变的患者应用PCR-DNA测序法对P19ink4d基因序列进行测定,分析有无突变。结果:噪声刺激后,各实验组听力与刺激前及对照组(未暴露噪声组)相比均明显下降;1d组较其他实验组下降更明显,7d与14d组间听力下降程度无明显差异。通过铺片检测发现随着时间的延长,外毛细胞排列发生混乱、变形以及缺损数量逐渐增加,内毛细胞未见明显缺失。噪声暴露3h后,外毛细胞P19ink4d蛋白免疫荧光染色明显增强,至噪声暴露后24h,P19ink4d蛋白的荧光染色强度基本恢复。通过Western blot检测发现噪声刺激后显示P19ink4d蛋白在3～6h出现暂时性的上调。本研究在400例极重度感音神经性聋患者中均未发现致P19ink4d基因的致病突变。结论:P19ink4d在耳蜗毛细胞上的表达在噪声刺激后显著增加,可能作为特异性基因在噪声性聋的发生、发展中起着重要作用。
Objective:This study aimed to investigate the P19 ink4 dexpression in cochlea of mice model with noise induced hearing loss and the role of P19 ink4 din the degeneration of inner ear cells.It also searched for P19 ink4 dgene alterations in patients with profound sensorineural deafness.Method:CBA/J mice were exposed to broad band noise at 101 dB SPL for 2 hours,auditory brainstem response(ABR)were examined to confirm noise lead to the permanent threshold shift.Immunohistochemical staining,Western blotting,and real-time polymerase chain reaction(PCR)were performed on cochlear tissues,to elucidate changes in P19 ink4 dexpression in mice after noise exposure.For clinical evaluation,400 children from unrelated families with severe or profound sensorineural hearing loss(SNHL)were recruited,genomic DNA was obtained from the patients and was subjected to DNA microarray to screen mutations in 4 most common genes.The sample that carried none of the common mutant alleles were subjected to PCR and sequenced to detect mutations in P19 ink4 dgene.Result:The ABR threshold shift of mice in the experimental group significantly increased after noise exposure and was higher than that in the null-noise group.The ABR of 1 day post noise was least among experimental groups and there is no statistical different between ABR of 7 days and 14 days post noise.The missing of outer hair cells occurred after noise exposure,while the inner hair cells hardly miss.It was found that the P19 ink4 dexpression increased significantly in the inner ear cells 3 hours after noise exposure,then recovered in 24 hours.Western blot indicated that the amount of P19 ink4 dincreased transitorily 3-6 hafter the noise.However,no mutation existed within the coding exons of P19 ink4 din the patients with profound sensorineural deafness.Conclusion:The results support the concept that P19 ink4 dmay play an important role in the pathogenesis and development of noise induced hearing loss.
Objective:This study aimed to investigate the P19 ink4 dexpression in cochlea of mice model with noise induced hearing loss and the role of P19 ink4 din the degeneration of inner ear cells.It also searched for P19 ink4 dgene alterations in patients with profound sensorineural deafness.Method:CBA/J mice were exposed to broad band noise at 101 dB SPL for 2 hours,auditory brainstem response(ABR)were examined to confirm noise lead to the permanent threshold shift.Immunohistochemical staining,Western blotting,and real-time polymerase chain reaction(PCR)were performed on cochlear tissues,to elucidate changes in P19 ink4 dexpression in mice after noise exposure.For clinical evaluation,400 children from unrelated families with severe or profound sensorineural hearing loss(SNHL)were recruited,genomic DNA was obtained from the patients and was subjected to DNA microarray to screen mutations in 4 most common genes.The sample that carried none of the common mutant alleles were subjected to PCR and sequenced to detect mutations in P19 ink4 dgene.Result:The ABR threshold shift of mice in the experimental group significantly increased after noise exposure and was higher than that in the null-noise group.The ABR of 1 day post noise was least among experimental groups and there is no statistical different between ABR of 7 days and 14 days post noise.The missing of outer hair cells occurred after noise exposure,while the inner hair cells hardly miss.It was found that the P19 ink4 dexpression increased significantly in the inner ear cells 3 hours after noise exposure,then recovered in 24 hours.Western blot indicated that the amount of P19 ink4 dincreased transitorily 3-6 hafter the noise.However,no mutation existed within the coding exons of P19 ink4 din the patients with profound sensorineural deafness.Conclusion:The results support the concept that P19 ink4 dmay play an important role in the pathogenesis and development of noise induced hearing loss.
引文
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[14]张志远,刘凯,陈燕红,等.miR-183家族在噪声性聋发生发展中的表达及研究[J].临床耳鼻咽喉头颈外科杂志,2014,28(7):468-472.
[15]SAMSON J,WIKTOREK-SMAGUR A,POLITANSKI P,et al.Noise-induced time-dependent changes in oxidative stress in the mouse cochlea and attenuation by D-methionine[J].Neuroscience,2008,152:146-150.
[16] HU B H,HENDERSON D,NICOTERA T M.Involvement of apoptosis in progression of cochlear lesion following exposure to intensenoise[J].Hear Res,2002,166:62-71.
[17]ZINDY F,CUNNINGHAM J J,SHERR C J,et al.Postnatal neuronal proliferation in mice lacking Ink4d and Kip1inhibitors of cyclin-dependent kinases[J].Proc Natl Acad Sci U S A,1999,96:13462-13467.
[2] HIRAI H,ROUSSEL M F,KATO J Y,et al.Novel INK4proteins,p19and p18,are specific inhibitors of the cyclin D-dependent kinases CDK4and CDK6[J].Mol Cell Biol,1995,15:2672-2681.
[3] CHEN F Q,ZHENG H W,HILL K,et al.Traumatic noise activates Rho-family GTPases through transient cellular energy depletion[J].J Neurosci,2012,32:12421-12430.
[4] SMITH I M,TURNBULL L W,SELLAR R J,et al.A modified screening protocol for the diagnosis of acoustic neuromas[J].Clin Otolaryngol Allied Sci,1990,15:167-171.
[5] SEBTI S M,DER C J.Opinion:searching for the elusive targets of farnesyl transferase inhibitors[J].Nat Rev Cancer,2003,3:945-951.
[6] CHEN P,ZINDY F,ABDALA C,et al.Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d[J].Nat Cell Biol,2003,5:422-426.
[7] LIU D X,GREENE L A.Neuronal apoptosis at the G1/S cell cycle checkpoint[J].Cell Tissue Res,2001,305:217-228.
[8] MA Q,GRATI M,BAI F,et al.Rescue from early-onset hearing loss in a mouse model lacking the cyclindependent kinase inhibitor p19Ink4d[J].Cell Death Dis,2016,7:e2131.
[9] THULLBERG M,BARTKOVA J,KHAN S,et al.Distinct versus redundant properties among members of the INK4family of cyclin-dependent kinase inhibitors[J].FEBS Lett,2000,470:161-166.
[10]HERRUP K,YANG Y.Pictures in molecular medicine:contemplating Alzheimer's disease as cancer:a loss of cell-cycle control[J].Trends Mol Med,2001,7:527-527.
[11]NGUYEN M D,MUSHYNSKI W E,JULIEN J P.Cycling at the interface between neurodevelopment and neurodegeneration[J].Cell Death Differ,2002,9:1294-1306.
[12]TAVERA-MENDOZA L E,WANG T T,WHITE J H.p19INK4Dand cell death[J].Cell Cycle,2006,5:596-598.
[13]CERUTI J M,SCASSA M E,FLOJ M,et al.Induction of p19INK4din response to ultraviolet light improves DNA repair and confers resistance to apoptosis in neuroblastoma cells[J].Oncogene,2005,24:4065-4080.
[14]张志远,刘凯,陈燕红,等.miR-183家族在噪声性聋发生发展中的表达及研究[J].临床耳鼻咽喉头颈外科杂志,2014,28(7):468-472.
[15]SAMSON J,WIKTOREK-SMAGUR A,POLITANSKI P,et al.Noise-induced time-dependent changes in oxidative stress in the mouse cochlea and attenuation by D-methionine[J].Neuroscience,2008,152:146-150.
[16] HU B H,HENDERSON D,NICOTERA T M.Involvement of apoptosis in progression of cochlear lesion following exposure to intensenoise[J].Hear Res,2002,166:62-71.
[17]ZINDY F,CUNNINGHAM J J,SHERR C J,et al.Postnatal neuronal proliferation in mice lacking Ink4d and Kip1inhibitors of cyclin-dependent kinases[J].Proc Natl Acad Sci U S A,1999,96:13462-13467.