腺病毒介导的Bcl-2基因对原代螺旋神经节细胞氧化应激损伤保护作用的研究

目的 研究腺病毒介导Bcl-2基因对于原代培养螺旋神经节细胞氧化应激损伤的作用.方法 大鼠螺旋神经节细胞原代培养,分AdEGFP加100 μmol/L H2O2作用组(A组)及AdEGFP/Bcl-2加100 μmol/L H2O2作用组(B组).hochest33258及PI染色,计数受损伤细胞的百分比.结果 100 μmol/L H2O2作用下细胞受损伤的方式主要以早期凋亡表现为主.B组中受损伤细胞的数目较A组明显减少,两组差异有显著性(P<0.01).结论 腺病毒介导Bcl-2蛋白表达可以抑制H2O2诱导螺旋神经节细胞凋亡的发生.     

腺病毒介导Bcl-2基因对原代螺旋神经节细胞存活的促进作用

目的研究外源性细胞凋亡相关基因Bcl-2对原代培养的螺旋神经节细胞(spiral ganglion cells,SGCs)存活及突起生长的影响,以期找到保护SGCs的有效方法。方法大鼠螺旋神经节细胞原代培养,免疫印迹法(western blot)检测转染AdEasy系统构建携带Bcl-2和增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)的腺病毒后SGCs表达Bcl-2蛋白的水平。免疫细胞组织化学染色鉴定螺旋神经节细胞,计数SGCs的数目。结果AdEGFP/Bcl-2组SGCs的存活数目分别高于AdEGFP组及空白对照组,AdEGFP组的存活SGCs细胞数略少于空白对照组。结论Bcl-2蛋白能促进体外SGCs细胞存活,腺病毒本身有轻微的细胞毒性,但表达外源性Bcl-2基因却可以保护SGCs不受腺病毒细胞毒性损害。     

哇巴因对大鼠耳蜗螺旋神经节细胞的损伤

目的 观察哇巴因(ouabain)对SD大鼠耳蜗螺旋神经节细胞(spiral ganglion cell,SGC)的损伤.方法 75只雄性SD大鼠随机数字表法分为5组,除健康对照组外,其余4组经耳蜗鼓阶钻孔分别给予0.01、0.02、0.05 mmol/L哇巴因以及生理盐水.术后7d行畸变产物耳声发射(DPOAE)及听性脑干反应(ABR)测试;大鼠处死后取耳蜗组织,通过免疫组化和透射电镜观察耳蜗SGC形态学变化.体外分离培养孕14d胎鼠SGC,加入1×10-8mmol/L哇巴因,通过光镜和扫描电镜观察哇巴因对离体培养SGC的损伤.结果 鼓阶注入哇巴因后,大鼠DPOAE无明显改变,各组之间不同频率DPOAE幅值差异均无统计学意义(P值均＞0.05).耳蜗鼓阶注入哇巴因后,大鼠ABR 反应阈升高,且升高幅度随着哇巴因浓度的增加而加大;哇巴因组大鼠ABR阈值与生理盐水组及健康对照组相比,差异具有统计学意义(P值均＜0.05).电镜下哇巴因组大鼠SGC发生退行性改变,细胞器结构破坏,核膜溶解,神经髓鞘崩解、空泡化.哇巴因同样可导致离体培养SGC的损伤,胞体皱缩、变小,突起缩短、断裂,细胞结构破坏.结论 无论是在体还是体外细胞培养,哇巴因均可确切造成SGC损伤.     

氧化还原因子1对体外培养大鼠耳蜗螺旋神经节氧化损伤的保护作用

目的 观察表达无嘌呤无嘧啶核酸内切酶/氧化还原因子1(apurinic/apyrimidimicendonuclase/redox factor 1,APE/Ref-1)的重组腺病毒感染对H2O2所致体外培养大鼠耳蜗螺旋神经节细胞氧化损伤的保护作用.方法 体外培养大鼠耳蜗螺旋神经节细胞,APE/Ref-1腺病毒表达载体感染48 h后,加入不同浓度H2O2(0、10、25、50、100及300 μmol/L)干预1 h,更换正常培养液后继续培养24 h,通过蛋白免疫印迹分析、四甲基偶氮唑蓝(MTT)法、原位缺口末端标记法(TUNEL)分别检测感染后螺旋神经节细胞APE/Ref-1蛋白表达、细胞活力以及凋亡情况.结果 通过腺病毒感染实现了APE/Ref-1基因在体外培养耳蜗螺旋神经节细胞的过表达,H2O2浓度为50～300 μmol/L时,APE/Ref-1组同对照组比较,细胞活力提高、凋亡率降低(P值均<0.01).结论 腺病毒介导的APE/Ref-1过表达对H2O2所致螺旋神经节细胞氧化损伤具有保护作用.     

哇巴因对小鼠内耳神经胶质细胞的影响

目的 研究哇巴因对小鼠内耳神经胶质细胞的影响,为干细胞移植治疗感音神经性聋的研究奠定基础.方法 成年雌性SPF级CBA/J小鼠60只,随机分为实验组和对照组,每组30只;实验组动物接受哇巴因(3 mM)经圆窗渗透给药,对照组给予等量生理盐水,于给药后7、14和30天用免疫组织荧光染色法观察位于耳蜗螺旋神经节内的内耳神经胶质细胞(inner ear glial cells,IEGs)的变化.结果 给药后7、14、30天实验组耳蜗各回螺旋神经节内可见内耳神经胶质细胞存活,但数量减少,排列紊乱;与对照组相比,哇巴因给药后7天实验组耳蜗各回神经胶质细胞的数量及密度即显著减少,给药后14天及30天后胶质细胞数量进一步减少;给药后30天各回螺旋神经节内内耳神经胶质细胞数降至最低,与实验组给药后7天、14天及与同时间点对照组比较显著减少,差异有统计学意义(P<0.05).结论 哇巴因经圆窗渗透给药可直接造成CBA/J小鼠内耳螺旋神经节内神经胶质细胞的急性进行性损伤.     

阳离子脂质体介导小鼠原代螺旋神经节细胞NT-3基因转染的实验研究

目的通过阳离子脂质体携带构建的神经营养因子-3(NT-3,Neurotrophin-3)-绿色荧光蛋白基因(pEGFPC2)转染小鼠耳蜗原代培养的螺旋神经节细胞,观察NT-3-pEGFPC2的共表达及其对螺旋神经节细胞生长的影响。方法构建携带绿色荧光蛋白报告基因的重组质粒-NT3 pEGFPC2,建立体外培养原代小鼠耳蜗螺旋神经节细胞的方法,并用神经丝蛋白(NF200)单克隆抗体进行免疫组化染色鉴定。利用阳离子脂质体携带重组质粒瞬时转染原代小鼠耳蜗螺旋神经节细胞,观察转染后NT-3的表达及对螺旋神经节细胞生长数量的影响。结果成功培养了小鼠原代耳蜗螺旋神经节细胞。基因转染后24 h在荧光显微镜下观察到胞体及轴突发出绿色荧光的螺旋神经节细胞。用Western blot方法检测到相应NT-3蛋白表达。继续培养12 d后各组细胞数量均减少,但NT-3转染组细胞减少数量低于空载体组。结论阳离子脂质体可作为NT-3基因的载体。NT-3的基因转染对抑制耳蜗螺旋神经节细胞的退行性变有一定作用。     

HO-1在顺铂诱导豚鼠螺旋神经元损伤中的作用

目的检测血红素加氧酶-1在豚鼠耳蜗螺旋神经节顺铂损伤后的表达变化。方法通过免疫组织化学及Western Blot技术,观察耳蜗螺旋神经元损伤后HO-1在不同组间的表达变化。将32只健康豚鼠随机分为4组,每组8只。A组对照组:按体重以0.9%生理盐水12ml/kg腹腔注射,早晚各一次,持续3天。B组顺铂组:按体重12mg/kg顺铂腹腔注射(IP),早晚各一次,持续3天。C组阿魏酸(FA)+顺铂组:先150mg/kg FA腹腔注射预处理1h,再以12mg/kg顺铂IP,早晚各一次,持续三天。D组FA+锌原卟啉Ⅸ(ZnppⅨ)+顺铂组:同时给予150mg/kg FA+ZnppⅨ15umol/kg IP预处理1h,再以12mg/kg顺铂IP,早晚各一次,持续三天。采用免疫组织化学及Wstern Blot技术检测不同组间螺旋神经元HO-1蛋白表达变化。结果在不同的分组中,HO-1在耳蜗螺旋神经节中表达量不同。空白组中,螺旋神经节组织存在微弱的HO-1表达,其蛋白表达量为0.48±0.07;A组蛋白表达量为0.81±0.07,B组蛋白表达量为0.9±0.05,C组蛋白表达量为0.61±0.07。4个组两两间差异均有统计学意义(p<0.0001)。结论 HO-1在C组中表达量最多,B组次之,D组最少,表明HO-1可能参与顺铂损伤螺旋神经元后的修复过程。     

MK-801拮抗水杨酸钠致螺旋神经节细胞损伤的实验研究

目的探讨非特异性阻断NMDA受体拮抗水杨酸钠对离体培养螺旋神经节细胞兴奋性损伤的作用。方法将培养的耳蜗螺旋神经节细胞随机分为3组,分别为正常对照组:培养液中仅加入1mM谷氨酸;水杨酸钠组:1mM谷氨酸+5mM水杨酸钠;MK-801组:50μM MK-801+1mM谷氨酸+5mM水杨酸钠。培养24h后加药物干预3h,收集细胞采用实时荧光定量PCR及免疫荧光技术检测BDNF exonⅣ,BDNF exonⅥ转录及Caspase-3mRNA转录和蛋白表达的改变,研究应用MK-801非特异性阻断NMDA受体拮抗水杨酸钠对离体培养螺旋神经节细胞兴奋性损伤的作用。结果水杨酸钠组较谷氨酸组及MK-801组螺旋神经节细胞中BDNF exonⅣ,BDNF exonⅥ和Caspase-3 mRNA的转录及Cas-pase-3蛋白的表达水平显著增高(P值均<0.05);BDNF exonⅣ的转录在MK-801组较谷氨酸组明显降低(P<0.05);BDNF exonⅥ的转录在MK-801组较谷氨酸组未发生具有统计学意义的改变;Caspase-3的转录及蛋白的表达在MK-801组较谷氨酸组显著提高(P<0.05)。结论非特异性阻断NMDA受体能拮抗水杨酸钠引起的离体培养螺旋神经节细胞中BDNFexonⅣ,BDNFexonⅥ及Caspase-3上调。     

人Bcl-2基因重组腺病毒的构建及其在螺旋神经节细胞中的表达

目的构建表达人Bcl-2基因的重组腺病毒，并研究其在原代培养螺旋神经节细胞(spiral ganglion cells，SGC)的表达特性。方法采用细菌内同源重组法，从pUC-CAGGS,／Bcl-2质粒中获得人Bcl-2 cDNA，克隆到腺病毒穿梭质粒pAdTrack-CMV上，后者和腺病毒骨架质粒pAdEasy-1在细菌内同源重组，在HEK293细胞中包装成为重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒，电镜观察病毒颗粒，用病毒感染原代培养的大鼠螺旋神经节细胞，在荧光显微镜下观察感染效率，用逆转录聚合酶链反应方法检测Bcl-2基因在螺旋神经节细胞的转录表达，用Western Blot检测其蛋白的表达。结果经酶切鉴定证实重组腺病毒质粒构建成功，电镜显示包装细胞中有大量病毒颗粒存在，呈规则六边形。荧光显微镜下观测到感染病毒的SGC细胞发出明亮的绿色荧光。分别在mRNA水平及蛋白水平证实有外源性Bcl-2基因的表达。结论细菌内同源重组法是一种简便、高效的制备方法。构建的重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒能有效的感染SGC，并可在SGC中正确地转录和翻译，为进一步研究腺病毒介导的Bcl-2基因对螺旋神经节损伤的保护作用奠定基础。     

腺病毒介导bcl-2基因转染对顺铂所致大鼠螺旋神经节细胞损伤的拮抗作用

目的 通过腺病毒(adenovirus,Ad)载体介导bcl-2基因转染体外培养的新生大鼠耳蜗螺旋神经节细胞(spiral ganglion cells,SGC),探讨bel-2蛋白过度表达对顺铂所致SGC损伤的拮抗作用.方法 体外培养新生大鼠SGC,携带绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒载体Ad-GFP转染SGC,并行神经丝蛋白(NF200)免疫细胞化学染色鉴定,激光共聚焦扫描荧光显微镜下观察.携带bel-2基因的腺病毒载体Ad-bel-2转染SGC,蛋白质印迹法(Western Blot)检测bcl-2蛋白表达.设立Ad-bcl-2转染加顺铂组(A组),Ad-GFP转染加顺铂组(B组),顺铂组(C组)和正常对照组(D组),顺铂作用浓度为2μg,/ml;顺铂作用48 h后,行各组SGC计数,并通过lmageJ软件测量各组SGC轴突长度.结果 成功分离并培养新生大鼠SGC.激光共聚焦扫描荧光显微镜下观察到腺病毒载体可安全高效转染体外培养的SGC.Ad-bcl-2转染3 d后,Western Blot检测有外源性人bcl-2基因的高效表达,而Ad-GFP转染组和顺铂组未检测到表达.顺铂作用后,A、B、C组部分SGC细胞突起变短、萎缩,胞体缩小、变圆,甚至浮起.A组SGC数目明显多于B组和C组(P值均＜0.01),但少于D组(P＜0.05);A组SGC轴突长度明显长于B组和C组,但短于D组(P值均＜0.01).结论 腺病毒能够安全高效地转染体外培养的新生大鼠SGC.bel-2蛋白过表达对顺铂所致SGC损伤有一定的拮抗作用.     

不同培养方法对螺旋神经元体外生长的影响

目的 观察不同培养条件下螺旋神经元体外生长情况,获得高纯度螺旋神经元培养物.方法 利用酶消化法、剪切分离结合酶消化法对出生后3～5天的大鼠螺旋神经节进行分离,分别在血清培养基和神经元专用培养基内培养,观察神经元形态及生长情况,并进行免疫组化鉴定.结果 神经元专用培养基比含血清培养基能够得到纯度更高的神经元,且细胞形态良好,轴突再生能力强.结论 酶消化结合剪切分离螺旋神经节,经神经元专用培养基培养,能获得高纯度的螺旋神经元.     

Peripheral and central target-derived trophic factor(s) effects on auditory neurons.

In the developing inner ear, a naturally occurring programmed cell death of cochleovestibular ganglion (CVG) neurons as well as peripheral and central target-derived trophic effects on survival of embryonic CVG neurons are known. To further analyze these target derived trophic interactions, spiral ganglion explants obtained from 5 day postpartum (P5) rat pups were cultured with an intact organ of Corti and in the absence of Corti's organ. Both neuronal survival and neurite extension were influenced by the presence of this peripheral target tissue. Local destruction of Corti's organ caused both neuritic retraction and neuronal cell death to occur in a corresponding portion of the spiral ganglion. This peripheral target-derived neurotrophic effect may be mediated by a diffusible factor(s) since organ of Corti conditioned medium also had a neurotrophic effect on the survival of auditory neurons in cell cultures of dissociated spiral ganglia from P5 rat pups. A component of central target tissue, i.e. astrocytes, was also shown to release a diffusible factor(s) that supported the survival of dissociated P5 rat spiral ganglion neurons. The neurotrophic effects on the in vitro survival of spiral ganglion neurons by both of these conditioned medium factors were concentration dependent.     

应用膜片钳技术对螺旋神经元放电类型的初步研究

目的：研究原代培养新生鼠螺旋神经元的放电类型。方法：应用膜片钳技术全细胞构型从原代培养的螺旋神经元获得电流钳记录。结果：从螺旋神经元记录到动作电位，螺旋神经元具有不一致的放电特征。结论：螺旋神经元具有快适应和慢适应两种放电特征，多数神经元为快适应神经元。     

ouabain-induced auditory nerve degeneration in congenic ly5.1 mice

The Ly5.1 mouse,also termed B6.SJL-Ptprca Pepcb/BoyJ,is a congenic strain widely used as a recipient in animal studies of bone marrow transplant.Our previous study documented that a majority of type Ⅰ ...     

Ouabain application to the round window of the gerbil cochlea: A model of auditory neuropathy and apoptosis

The physiological and morphological changes resulting from acute and chronic infusion of ouabain onto the intact round-window (RW) membrane were examined in the gerbil cochlea. Osmotic pumps fitted with cannulas allowed chronic (0.5-8 days) infusions of ouabain. Acute and short-term applications of ouabain (1-24 h) induced an increase in auditory-nerve compound action potential (CAP) thresholds at high frequencies with lower frequencies unaffected. The resulting threshold shifts were basically all (no response) or none (normal thresholds), with a sharp demarcation between high and low frequencies. Survival times of 2 days or greater after ouabain exposure resulted in complete auditory neuropathy with no CAP response present at any frequency. Distortion product otoacoustic emissions (DPOAEs) and the endocochlear potential (EP) were largely unaffected by the ouabain indicating normal function of the outer hair cells (OHC) and stria vascularis. One to 3 days after short-term applications, apoptosis was evident among the spiral ganglion neurons assessed both morphologically and with TdT-mediated dUTP-biotin nick end labeling (TUNEL). With 4-8 day survival times, most spiral ganglion cells were absent; however, a few cell bodies remained intact in many ganglia profiles. These surviving neurons had many of the characteristics of type II afferents. Our working hypothesis is that the ouabain induces a spreading depression among the type I ganglion cells by blocking the Na,K-ATPase pump. Because of the constant spike activity of these cells, the ouabain rapidly alters potassium concentrations within ([K+]i) and external to ([K+]o) the ganglion cells, thereby initiating an apoptotic cascade.     

Inhibition of the c-Jun N-terminal kinase signaling pathway influences neurite outgrowth of spiral ganglion neurons in vitro

OBJECTIVES: Inhibitors of the c-Jun N-terminal kinase (JNK) signaling pathway have been demonstrated to protect hair cells of the auditory system and different types of neurons from various insults, and their use for future therapeutic applications has been proposed. In the study, we evaluated the effects of inhibition of the JNK pathway on process outgrowth from spiral ganglion neurons. METHODS: Spiral ganglion explants from rats (postnatal days 3-5) that were cultured on laminin were treated with neurotrophin-3 and/or the JNK signaling pathway inhibitor CEP-11004. Both neurite length and number of the explants were evaluated and statistically analyzed by analysis of variance. RESULTS: Inhibition of the JNK signaling pathway reduced process outgrowth from spiral ganglion explants. The reduction, both in length and number of neurites, was reversed by the application of neurotrophin-3. CONCLUSIONS: The results indicate that an intact JNK signaling pathway is important for process outgrowth of spiral ganglion neurons. However, neurotrophin-3 stimulates process extension by a JNK independent pathway. Our results demonstrate that inhibition of the JNK pathway can have adverse effects on the extension of spiral ganglion neurons, but that the negative effects can be ameliorated by appropriate treatment.     

Study on voltage-sensitive current of spiral ganglion cells in mice organ of Corti culture]

OBJECTIVE: To investigate the property of voltage-sensitive current in cochlear spiral ganglion cells of the C57BL/10J mice, an inbred strain which develops early onset hearing loss. METHODS: Organotypic cultures of organ of Corti were prepared from neonatal mice 0-5 days of age. Whole-cell current and voltage clamp techniques were used to study Na+, K+ and Ca2+ currents of the spiral ganglion cells in culture. RESULTS: Cultures were maintained for 8-48 hours before use. Ganglion cells were identified first through their anatomical positions and finally through fast negative Na+ current. Spontaneous action potentials were recorded from some ganglion cells (4 out of 39). When present, spontaneous rates were around 20 spikes/sec, and might be as high as 135 spikes/sec. The mean resting potential was (-55 +/- 5) mV (n = 39). Under voltage clamp conditions, transient inward currents (negative) and outward (positive), steady-state voltage-dependent currents were recorded in normal HBSS. Rapid inward currents were totally blocked by 300 nM TTX applied locally to the culture. Inward currents recovered quickly after TTX wash out suggesting that the transient inward current was carried by Na+. The mean maximum amplitude of Na+ current was (-2.0 +/- 1.1) nA (n = 39) recorded in HBSS. Adding TEA (10 mmol/L) and 4-AP (0.15 mmol/L) to the bath solution or replacing K+ with Ca+ in the pipette solution partly blocked the sustained outward current. This suggests that the outward current was carried by K+. The mean maximum amplitude of K+ was (3.0 +/- 1.3) nA (n = 39) with 140 mM K+ in the pipette. Inward Ca2+ current was recorded in Ba2+ solution which mean peak amplitude was (-1.0 +/- 0.7) nA (n = 20). Ca2+ currents were reversibly blocked by 100 microM Cd2+. CONCLUSION: Whole cell recordings from spiral ganglion neurons can be obtained from organotypic cultures of the organ of Corti. Fast Na+ current, sustained K+ current and L-type Ca2+ current were recorded in the spiral ganglion cells cultured for 1-2 days. Whole cell recording showed that cochlea spiral ganglion cells can generate spontaneous action potential one day after birth and the firing rates could reach levels equal to those recorded in vivo.     

Exogenous BDNF rescues rat spiral ganglion neurons in vivo.

OBJECTIVE: To determine if exogenous neurotrophins can prevent spiral ganglion neuron degeneration in the rat cochlea. BACKGROUND: The loss of hair cells resulting in sensorineural hearing loss also leads to the secondary degeneration of spiral ganglion neurons. The effectiveness of cochlear implantation in patients with profound sensorineural hearing loss relies in part on the survival of spiral ganglion neurons; therefore, any therapy that can prevent or halt the loss of these neurons would be of potential clinical benefit. Previous research has shown that intracochlear infusion with neurotrophins can provide trophic support to SGNs in deafened guinea pigs. Whether this effect is seen in other species remains to be determined. METHODS: After documenting the rate of spiral ganglion neuron degeneration after ototoxic deafening, we investigated the trophic effects of exogenous brain-derived neurotrophic factor (BDNF) on rat spiral ganglion neurons. The left cochleae of profoundly deafened rats were implanted with a drug delivery system connected to a mini-osmotic pump. BDNF or artificial perilymph was infused for 28 days; then the cochleae were prepared for histological study. RESULTS: Treatment with BDNF led to a statistically significant increase in spiral ganglion neuron density and a highly significant increase in spiral ganglion neuron soma area compared with artificial perilymph-treated and untreated deafened cochleae. CONCLUSION: The study has demonstrated the trophic advantage of exogenous BDNF in the mature rat cochlea and provides confidence that spiral ganglion neuron rescue after sensorineural hearing loss with exogenous BDNF may have clinical application.