【作者】 冯爽；

【导师】 苏纪平；

【作者基本信息】 广西医科大学 ， 耳鼻咽喉头颈外科， 2012， 博士

【摘要】 耳聋在耳鼻咽喉头颈外科常见病。由于滥用抗生素、噪声的污染、人口老龄化及高危遗传的婚姻等诸多因素,我国难治性感音神经性耳聋发病率正逐年增加。耳聋已成为我国第一致残疾病。因此防治耳聋,已成为临床上迫切需要解决的顽症之一。Src家族是一种非受体型蛋白酪氨酸激酶,它通过催化底物酪氨酸残端的磷酸化反应,将ATP的磷酸基团转移到底物,从而改变底物的构象及活性。Src家族共有9个成员,广泛分布于全身各组织和细胞中。Src家族成员都有相似的结构,如共有6个功能结构域,其中C-末端的酪氨酸Tyr527和激酶区活化环的Tyr416在调节Src家族酶活性过程中起着关键的作用。已知在中枢神经系统,Src家族参与调节神经系统发育、迁徙、突触可塑性等生理过程,并介导缺血性损伤等兴奋毒性作用,如Src家族可通过磷酸化与细胞迁徙相关的蛋白,调控神经元在发育过程中的迁徙过程；Src家族成员Src可通过调控兴奋性受体NMDA受体磷酸化水平,促进长时程增强或长时程抑制的产生,调节突触可塑性,这被认为与癫痫、阿茨海默氏病等神经系统疾病的发病机制有关。有研究发现抑制蛋白酪氨酸激酶Src家族可有效缓解噪声引起的的耳蜗损害。因此,Src家族可能参与耳聋的病理生理过程。听觉信息在耳蜗转换为听神经动作电位,传向中枢。听神经动作电位发放异常,可导致听力下降。动作电位的产生主要取决于电压门控性钠通道和钾通道的开放。目前已明确调控听神经元电压门控性离子通道磷酸化水平,可影响听神经动作电位发放。研究虽已证实Src家族可参与调控电压门控性钠通道和钾通道,但对于Src家族在神经系统直接调控钠通道和钾通道的机制,目前并不明确；尤其是在听觉神经系统的研究,尚处于空白。耳蜗螺旋神经节神经元(SGN)是初级听觉神经元,是连接外周毛细胞和听觉中枢的重要枢纽,听神经动作电位起于SGN。已知SGN常是噪声和耳毒性药物等兴奋毒性作用的靶点。SGN的电压门控性钠通道和钾通道开放异常可影响听力。因此在本实验中,通过细胞原代培养技术,建立SGN体外培养模型；利用全细胞记录膜片钳技术,观察内源性Src家族被抑制后对SGN电压门控性钠通道和钾通道的作用特点,明确Src家族是否参与调控听神经系统动作电位的传导。通过观察Src家族各种调节因子对SGN电压门控性钠通道和钾通道的I-V曲线,钠电流宏观激活和失活(macroscopic activation and inactivation),电导,激活曲线,稳态失活曲线和失活后恢复曲线等电生理学特性的作用,明确Src家族在调控听神经动作电位过程的作用机制。最后观察Src家族代表性成员Src对SGN钠通道和钾通道的作用。通过本实验,将有利于揭示Src家族在听神经动作电位传播过程所扮演的角色,也为临床上耳聋防治提供新的理论指导。实验内容包括以下三部分：第一部分：Src家族对螺旋神经节神经元电压门控性钠通道和钾通道的调控研究目的：观察蛋白酪氨酸激酶Src家族对原代培养的大鼠螺旋神经节神经元(SGN)电压门控性钠通道和钾通道电生理学性质的作用,探讨Src家族对SGN听神经动作电位产生过程的影响。研究方法：将SGN从耳蜗中分离出并原代培养,.24h后结合免疫荧光染色技术,首先利用抗N-200抗体和Hoechst3342鉴定SGN,然后用抗Src家族抗体,明确Src家族在SGN的表达。采用全细胞记录膜片钳技术,证实去极化刺激引出的内向和外向电流分别为电压门控性钠通道电流和电压门控性钾通道电流。Src家族被抑制后,观察同时记录的SGN电压门控性钠通道和钾通道电流的变化,明确Src家族对钠通道和钾通道的作用特点。再用无钠(NMDG)细胞外液置换正常NaCl细胞外液,观察Src家族被抑制后,对SGN电压门控性钾电流和1-V曲线的影响,明确Src家族对SGN钾通道作用。结果：(1)抗N-200抗体特异标记SGN胞膜,Hoechst3342可特异标记SGN胞核,证实从耳蜗分离出来的细胞为SGN。利用抗Src家族抗体,证实Src家族在SGN表达。(2)TTX和CsCl可分别阻断由去极化刺激引出的内向电流和外向电流,证实内向电流为电压门控性钠通道电流(工Na),外向电流为电压门控性钾通道电流(IK)。(3)10μM PP2作用后,抑制SGN的INa,INa为作用前59.6±15.7%(n=9, P <0.05);2μM SU6656作用后也抑制INa,INa为作用前68.6±7.5%(n=5,P<0.05)。而对照物10μM PP3和对照溶液作用后不影响SGN的INa。(4) PP, PP3, SU6656以及对照溶液均不影响同时记录的IK。(5)在NMDG细胞外液条件下,PP2, SU6656及对照溶液不影响钾通道I-V曲线的反转电位,也不影响IK。结论：Src家族抑制后可抑制SGN电压门控性钠通道电流,但对电压门控性钾通道作用不明显。第二部分：Src家族对螺旋神经节神经元电压门控性钠通道的调控及其电生理学机制研究目的：观察Src家族在抑制和激活条件下,对SGN电压门控性钠通道电生理学特性的影响,明确Src家族调控钠通道机制。研究方法：通过近距离给药方式,观察Src家族抑制剂PP2和SU6656,以及PP2拟合物PP3对SGN电压门控性钠通道电流(INa),I-V曲线,钠电流宏观激活和失活,电导,激活曲线,稳态失活曲线和失活后恢复曲线的作用。Src家族激活剂EPQ(pY)EEIPIA及其对照物则通过细胞内给药途径,同样观察Src家族激活后,SGN钠通道电流,I-V曲线及动力学性质的变化。结果：(1)10μMPP2作用后抑制SGN的INa,INa为给药前59.7±10%(n=7, P＜0.05)。2μM SU6656作用后也抑制INa,INa为给药前62±8.8%(n=7,P<0.05)。而1011M PP3和对照溶液不影响INa。(2)PP2和SU6656不影响SGN INa的宏观激活和失活。(3)PP2和SU6656作用后均抑制钠通道的I-V曲线峰值,但不影响1-V曲线钠通道反转电位。而PP3不影响I-V曲线。(4)PP2作用前后,SGN钠通道的电导值分别为(6.7±1)nS和(5.2±0.7)nS(n=7,P<0.05)；而SU6656作用前后,SGN钠通道的电导值(11±2.4)nS和(7.9±1.9)nS(n=7,P<0.05)。PP2和SU6656均抑制SGN钠通道的电导。PP3不影响对SGN INa的电导。(5)PP2和PP3, SU6656均不影响钠通道激活曲线。(6)PP2作用前后,钠通道的半失活电压(V1/2)分别为(-69.4±0.8)mV和(-77.2±0.5)mV,斜率κ分别为(9.5±0.4)和(9.6±0.4)(n=11,P<0.05),PP2使钠通道的稳态失活曲线移向超极化方向。SU6656作用前后,V1/2分别为(-64.6±0.3)mV和(-70.1±0.4)mV,κ分别为(7.1±0.3)和(7.7±0.4)(n=9,P<0.05),SU6656也使钠通道的稳态失活曲线移向超极化方向。PP3不影响INa的稳态失活曲线。(7)PP2作用前后,钠通道失活后恢复过程的时间常数分别为(7.8±4.5)ms和(17.4±5.9)ms(n=6,P<0.05),SU6656作用前后,钠通道失活后恢复过程的时间常数分别为(5±2.8)ms和(11.2±2.2)ms(n=6,P<0.05)。PP2和SU6656均延缓失活恢复过程。PP3不影响钠通道失活后恢复过程。(8) EPQ(pY)EEIPIA(1mM)作用后增强INa，INa为作用前131.3±5.2%(n=12,P<0.05)。对照物EPQYEEIPIA(1mM)和空白对照不影响INa。(9) EPQ(pY)EEIPIA不影响SGN INa的宏观激活和失活。(10) EPQ(pY)EEIPIA作用后增强钠通道的I-V曲线峰值。EPQ(pY)EEIPIA和EPQYEEIPIA均不影响钠通道的I-V曲线反转电位。(11)EPQ(pY)EEIPIA作用后增强SGN钠通道的电导,作用前后钠通道的电导值分别为(8.2±1.3)nS和(9.7±1.4)nS(n=9, P＜0.05)。EPQYEEIPIA不影响SGN钠通道的电导。(12) EPQ(pY)EEIPIA作用前后,钠通道半激活电压(V1/2)分别为(-36.4±0.3)mV和(-40.2±0.4)mV,斜率K为(3.5±0.3)和(3.4±0.4)(n=9,P<0.05),钠通道的激活曲线移向超极化方向。EPQYEEIPIA不影响钠通道的激活曲线。(13) EPQ(pY)EEIPIA和EPQYEEIPIA均不影响钠通道的稳态失活曲线。(14) EPQ(pY)EEIPIA作用前后,SGN钠通道的失活后恢复时间常数分别为(5.6±2.5)ms和(6.1±2.8)ms(n=10, P＞0.05)。EPQ(pY)EEIPIA不影响钠通道的失活后恢复过程。结论：Src家族的激活可上调SGN电压门控性钠通道的功能。Src家族不仅影响钠通道的电导,而且同时作用于钠通道的激活区和失活区,影响其激活过程和失活过程。Src家族对钠通道激活过程和失活过程的影响,与其活性水平有关。第三部分Src家族成员Src对螺旋神经节神经元电压门控性钠通道的作用研究目的：观察Src对SGN电压门控性钠通道电生理特性的影响,明确Src家族单个成员在调节SGN电压门控性钠通道过程中所起的作用。研究方法：利用全细胞记录膜片钳技术,通过细胞内给药途径,观察Src特异抑制剂Src40-58对SGN INa,钠电流宏观激活和失活,I-V曲线,电导,激活曲线,稳态失活曲线以及失活后恢复曲线的作用。并观察PP2预处理后,Src40-58对SGN INa作用的变化。结果：(1) Src40-58(0.3mg/ml)抑制SGN INa,INa为给药前73.8±3.5%(n=10,P<0.05)。Src40-58还抑制I-V曲线峰值,但不影响反转电位。(2)Src40-58不影响SGN INa的宏观激活和失活。(3)Src40-58抑制SGN钠通道的电导,作用前后钠通道的电导分别为(12±2.5)nS和(10±3)nS(n=7,P<0.05)。(4)Src40-58作用前后,钠通道半激活电压(V1/2)分别为(-38.5±0.4)mV和(-44.3±0.6)mV,斜率K为(3.8±0.4)和(3.2±0.5)(n=7,P<0.05)；而钠通道半失活电压(V1/2)分别为(-63.4±0.4)mV和(-67.9±0.5)mV,斜率K为(6.54±0.4)和(7.36±0.4)(n=9,P<0.05),钠通道的激活曲线和失活曲线均移向超极化方向。(5)Src40-58影响SGN钠通道的失活后恢复曲线。Src40-58作用前后,钠通道的失活后恢复时间常数分别为(1.1±0.5)和(1.7±0.7)ms(n=6, P<0.05), Src40-58延缓SGN钠通道的失活后恢复过程。(6)10μM PP2预处理5min,然后给予Src40-58,SGN INa为给药前96.5±11.7%(n=10,P>0.05),此时Src40-58不影响INa的大小,也不影响I-V曲线峰值和反转电位。(7)PP2预处理后可阻断Src40-58对SGN钠通道的电导的抑制作用,作用前后钠通道的电导分别为(8±0.9)nS和(7.5±1.2)nS(n=7,P>0.05)。(8)同理,PP2预处理后,Src40-58作用前后SGN钠通道的半失活电压(V1/2)分别为(-71.2±0.4)mV和(-73.3±0.4)mV,斜率K为(6.7±0.4)和(7.1±0.3)(n=8,P>0.05),稳态失活曲线未出现明显位移。而半激活电压(V1/2)分别为(-33.8±0.5)mV和(-39.9±1)mV,斜率K为(3.7±0.7)和(4.8±0.8)(n=9,P<0.05),钠通道的激活曲线仍移向超极化方向。(9)同理,PP2预处理后阻断Src40-58对SGN钠通道的失活后恢复曲线的影响。Src40-58作用前后,钠通道的失活后恢复时间常数分别为(7±4.6)和(7.4±4.6)ms(n=6,P>0.05)。结论：Src对SGN钠通道的具有与Src家族类似的抑制作用,主要通过抑制电导大小,增强其电压依赖性稳态失活过程,延缓失活后恢复过程。更多还原

【Abstract】 Hearing loss is common in Otolaryngology. It is about10%of population suffer from different degrees of dearness in developed countries. The incidence of deafness in china is increasing also year by year because of these factors such as the abuse of antibiotics, noise pollution, population aging and genetic risk factors such as marriage. The prevention of hearing loss has become to be urgent to address in clinic. It is shown that the inhibition of tyrosine protein kinase, Src family kinase (SFKs), could prevent the cochlear damage from noise induced hearing loss. It is implied Src family may be involved in the pathological procedure of hearing loss.SFKs is a family of non-receptor kinase that includes nine members. SFKs is able to catalyse the transfer of phosphate from ATP to a tyrosine residue of specific cell protein targets, and modify the molecular structure and activity of substrate. The activity of SFKs is regulated by the phophosrylation of tyrosine527in C terminus and tyrosine416in activation loop of kinase domain. It is invoved in the regulation of neurons development, migration, survival and synaptic plasticity, and also mediated the excitotoxicity process in nervous system, which may be the pathological mechanism of epilepsy, pain, Alzheimer’s disease and others neurodegenerative disease.Auditory information is converted as action potential in cochlear and then transmitted to the auditory central. Abnormal firing of auditory neurons can be lead to hearing loss. It is well known the voltage-gated sodium channel and potassium channels play critical roles in generation of action potentials. Some studies have indicated that the phosphorylated voltage-gated ion channels in auditory neurons would influence the firing of action potential. In addition, the voltage gated sodium and potassium channels are able to be regulated by SFKs in vivo. However, it is still unclear the effect of SFKs on sodium and potassium channels expressed in neurons, especially in auditory neurons. The cochlear spiral ganglion neurons (SGN) are primary auditory neurons, which interact with hair cells in peripheral and with auditory center in central. SGN is the origin of auditory action potential. Usually SGN is the target of excitotoxicity of noise or some ototoxic drugs. Dysfunction of voltage-gated sodium channels and potassium channels has proved to be induced hearing loss. Therefore, SGN cultured model in vivo was well established first by combination of cell primary culture technique and immunofluorescence staining in this study. The effect of endogenous SFKs on SGN voltage-gated sodium channel and potassium channel currents was investigated by whole cell configuration patch clamp technique. We also tested the regulation of I-V curve, activation curve and steady state inactivation curve of sodium channels and potassium channels by SFKs activator and inhibitor. To identify the role of Src in modification of action potential generated in SGN, we detected the influence of Src specific inhibitor Src40-58on the currents, macroscopic activation and inactivation, I-V curve, conductance, activation curve, steady state inactivation curve, recovery of INa on sodium and potassium channels. It will help us to illustrate the mechanism of cochlear impaired mediated by Src family, and provide a new guideline about deafness theorapy. This study is included three chapters:Chapter1:The regulation of voltage-gated sodium and potassium channels in spiral ganglion neurons by endogenous Src family kinaseObjective:To understand the mechanism of regulation of action potential generation by SFKs, the effect of voltage-gated sodium and potassium channels in spiral ganglion neurons (SGN) by SFKs inhibitors was detected.Methods:SGNs were dissected from cochlear tissue and plated into culture dishes. SGNs were identified by immunofluorescence staining. The location of SFKs in SGN was detected also. Inward and outward currents were indentified by TTX and Cs". To testify the effect on sodium channes and potassium channels in SGN by SFKs, the voltage-gated sodium and potassium channels currents were tested by whole cell configuration patch clamp technique after applied PP2, PP3, SU6656and vehicle. Subsequently, Na+in the extracellular solution was replaced with N-methy-D-Glucomine (NMDG) to block Na+currents. To investigate the regulation of Src kinase on voltage-gated potassium channel, potassium currents and Ⅰ-Ⅴ curve were detected when SFKs was applied.Results:(1) N-200selectively interacted with the membrane of SGN, and Hoechst3342selectively interacted with the nucleus of SGN. SFKs were detected in SGN by anti-SFKs antibody.(2) TTX and Cs+could block the inward currents and outward currents induced by depolarized pulse.(3) The sodium currents were significantly inhibited after applied10μM PP2or2μM SU6656. The currents is59.6±15.7%(n=9, P<0.05) and68.6±7.5%(n=5, P ＜0.05) compared with those recorded before SFKs inhibitors application. No significant change in the currents was found following application of PP3(10μM) or vehicle.(4) No change in potassium currents recorded at the same time was induced following application of PP2, PP3, SU6656or vehicle.(5) Potassium currents were recorded with Na+free extracellular solution in which Na+was replaced with NMDG. No change in potassium currents and I-V curve was detected following application of PP2, SU6656or vehicle.Conclusions:The inhibition of SGN voltage-gated sodium channel is induced by SFKs inhibitors. There is no change on potassium currents after inhibited SFKs. Chapter2:The regulation of kinetic of voltage-gated sodium channels in spiral ganglion neurons by endogenous Src family kinaseObjective:To investigate the mechanism of regulation on kinetic properties of voltage-gated sodium channels in SGN by SFKs.Methods:K+in the solutions was replaced with Cs+to block potassium currents. PP2. PP3. SU6656were diluted into extracellular solution, and Src activated peptide EPQ(pY)EEIPIA and control peptide EPQYEEIPIA were diluted into intracellular solution. The sodium currents, macroscopic activation and inactivation. I-V curve, conductance, activation curve, steady state curve and recovery of inactivation were tested following applied SFKs inhibitors or activators. Results:(1) The sodium currents were significantly reduced when applied10μM PP2or2μM SU6656. The currents were59.7±10%(n=7, P＜0.05) and62±8.8%(n=7, P＜0.05) respectively, when compared with those recorded before SFKs inhibitors application. No significant change in the sodium currents was found following application of10μM PP3or vehicle.(2) PP2and SU6656did not affect the macroscopic activation and inactivation.(3) Application of PP2or SU6656significantly reduced peak Na+current density in I-V curve without changing reversal potential. PP3or vehicle application did not produce such changes.(4) PP2reduced the conductance of sodium channel in SGN (control:6.7±1nS; PP2:5.2±0.7nS, n=7, P＜0.05). SU6656also reduced the conductance of sodium channel (control:11±2.4nS; SU6656:7.9±1.9nS, n=7, P＜0.05). PP3did not change the the conductance of sodium channel.(5) No change in activation curve of sodium channel was induced following application of PP2, PP3and SU6656.(6) The steady state inactivation curve was shifted to the left after PP2application (control:V1/2=-69.4±0.8mV, k=9.5±0.4; PP2:V1/2=-77.2±0.5mV, k=9.6±0.4, n=11,P＜0.05). Similarly, the steady state inactivation curve was also shifted to the left after application of SU6656(control:V1/2=-64.6±0.3mV, k=7.1±0.3; SU6656:V1/2=-70.1±0.4mV, k=7.7±0.4, n=9, P＜0.05). No significant change in the steady state inactivation curve was found following application of PP3.(7) The time constant of recovery of sodium channel was reduced by PP2(control:7.8±4.5ms; PP2:17.4±5.9ms; n=6, P＜0.05). The time constant of recovery of sodium channel was also reduced by SU6656(control:5±2.8ms; SU6656:11.2=2.2ms, n=6, P＜0.05). PP3did not affect the time constant of recovery. (8) The sodium currents were significantly potentiated when applied EPQ(pY)EEIPIA (1mM). The currents is131.3±5.2%(n=12, P＜0.05) comparing with the currents recorded immediately after the breakthrough. No significant change in the sodium currents was found following application of EPQYEEIPIA (1mM) or control pipette solution.(9) EPQ(pY)EEIPIA did not influence the macroscopic activation and inactivation.(10) Application of EPQ(pY)EEIPIA significantly increased peak Na+current density in I-V curve. EPQ(pY)EEIPIA did not change reversal potential. EPQYEEIPIA did not produce such changes.(11) EPQ(pY)EEIPIA inceased the conductance of sodium channel in SGN (control:8.2±1.3nS; EPQ(pY)EEIPIA:9.7±1.4nS, n=9, P＜0.05). EPQYEEIPIA did not produce such changes.(12) The activation curve was shifted to the left after EPQ(pY)EEIPIA application (control:V1/2:-36.4±0.3mV, k=3.5±0.3; EPQ(pY)EEIPIA:V1/2=-40.2±0.4mV, k=3.4±0.4, n=7, P＜0.05). No significant change in the activation curve was found following application of EPQYEEIPIA.(13) No change in activation curve of sodium channel was induced following application of either EPQ(pY)EEIPIA or EPQYEEIPIA.(14) The time constant of recovery of sodium channel was reduced by EPQ(pY)EEIPIA (control:5.6±2.5ms; EPQ(pY)EEIPIA:6.1±2.8ms, n=10, P＞0.05). EPQYEEIPIA did not produce such changes.Conclusions:Src family kinase regulates the voltage-gated sodium channels activtity in SGN. The conductance, activation and the steady-state inactivation of sodium channels are likely subjects to SFKs regulations. The effect on activation and inactivation of sodium channels is depended on the enzyme activity. Chapter3:The effect of Src on voltage-gated sodium channels in spiral ganglion neuronsObjective:To testify the regulation on voltage-gated sodium channel in spiral ganglion neurons by Src.Methods:Src40-58was diluted into intracellular solution. The sodium currents, macroscopic activation and inactivation, I-V curve, conductance, activation curve, steady state inactivation curve and recovery of INa were detected by whole cell configuration patch clamp technology. The effect of Src40-58on sodium currents in SGN was tested followed PP2pre-treated.Results:(1) The sodium currents were significantly reduced when applied Src40-58(0.3mg/ml). The currents were73.8±3.5%(n=10, P＜0.05) compared with those recorded before Src40-58application. Src40-58also reduced peak Na+current density in I-V curve without changing reversal potential.(2) Src40-58did not affect the macroscopic activation and inactivation.(3) Src40-58reduced the conductance of sodium channel in SGN (control:12±2.5nS:Src40-58:10±3nS. n=7. P＜0.05).(4) The steady state inactivation curve was shifted to the left following application of Src40-58(control:V1/2=-63.4±0.4mV, k=6.5±0.4; Src40-58:V1/2=-67.9±0.5mV,k=7.4±0.4, n=9, P＜0.05). Moreover, the activation curve was shifted to the left following application of Src40-58(control:V1/2=-38.5±0.4mV, k=3.8±0.4; Src40-58:V1/2=-44.3±0.6mV,k=3.2±0.5, n=7, P＜0.05).(5) The time constant of recovery of sodium channel was reduced by Src40-58(control:1.1±0.5ms; Src40-58:1.7±0.7ms, n=6, P＜0.05).(6) The inhibiton of Src40-58on the amplitude of sodium channel in SGN could be prevented in SGN bathed with solution with PP2(96.5±11.7%, n=10, P＞0.05). In this case, No change on peak Na+current density in I-V curve in SGN was found either.(7) The reduction on the conductance of sodium channel in SGN by Src40-58was prevented by PP2pre-treatment (control:8±0.9nS; Src40-58:7.5±1.2nS, n=7, P＞0.05).(8) The shift on steady state inactivation curve induced by Src40-58in SGN was prevented following PP2pre-treatment (control:V1/2=-71.2±0.4mV, k=6.7±0.4; Src40-58:V1/2=-73.3±0.4mV, k=7.1±0.3, n=8, P＞0.05). However, following application of Src40-58, the activation curve was still shifted to the left in SGN when was pre-treated by PP2(control:W1/2=-33.8±0.5mV, k=3.7±0.7; Src40-58: V1/2=-39.9±1mV, k=4.8±0.8, n=9, P＜0.05).(9) Following application of Src40-58, no change on the time constant of recovery of sodium channel was found in SGN bathed with PP2(control:7±4.6ms:Src40-58:7.4±4.6ms; n=6, P＞0.05).Conclusions:Src is involved in the regulation of sodium channels in SGN. The effect of Src on sodium channels in SGN is similar as that of SFKs更多还原