【作者】 李树峰；

【导师】 王正敏；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 第一部分螺旋神经元神经突电场导向生长研究体外模型的建立目的:建立适合的螺旋神经元神经突电场导向生长研究的体外模型体系,用于观察不同类型的电场对体外培养螺旋神经元神经突导向生长作用的研究。方法:出生4-6天的Sprague-Dawley大鼠,解剖出蜗轴,分别进行组织块培养和消化分离培养,铺片方法采取单纯层粘连蛋白(Laminin)、单纯多聚赖氨酸(Poly-D-lysine,PDL)、多聚赖氨酸加层粘连蛋白(PDL+Laminin)包被、单纯鼠尾胶原蛋白包被和不包被等5种方法,观察螺旋神经的神经突的生长情况。使用35mm培养皿,分别导入铂铱合金丝、环状电极和人工耳蜗接受刺激器的极阵构建电场,外接稳压电源、函数信号发生器和电荷平衡式双相脉冲电流产生装置。采用直接接种和刺激前后拍照与先培养再移植到活细胞长时间观察记录系统中连续监测的两种方法,记录生长锥的移动轨迹。结果:螺旋神经元组织块培养较消化分离培养更容易获得生长方向上无干扰的“自由”神经突。Laminin、PDL和两者联合铺片的培养体系中,神经突长出数量较多、生长情况较好。鼠尾胶铺片的培养体系中,非神经细胞生长过于旺盛。不铺片的培养体系中神经突很少长出,组织块贴壁率也较低。两种观察记录方法均能够实现神经突导向生长的动态观察研究。但直接接种的方法实验效率较低,且不易进行电场内的定位研究。培养后移植到活细胞长时间观察记录系统中连续监测的方法,能够在电场中任意定位神经突,获得生长锥的连续移动轨迹,培养环境稳定,有利于导向生长的研究。结论:Laminin、PDL或两者联合铺片结合组织块培养的方法更容易获得适合螺旋神经元神经突导向研究的“自由神经突”。移植培养观察可以实现高效的定位研究;活细胞长时间观察记录系统中连续监测的方法有利于维持稳定的培养环境和实现神经突生长的动态观察。第二部分稳态和脉冲直流电场对体外培养螺旋神经元神经突生长方向的影响目的:观察稳态和脉冲直流电场对体外培养螺旋神经元神经突生长方向的影响。方法:分别采用Laminin或PDL包被玻片,用组织块法体外培养螺旋神经元。选择有“自由神经突”形成的玻片移植到活细胞观察系统中,使神经突主干末段与平行电极电场体系中的电力线垂直。在不施加电场、100mV/mm的稳态直流电场和占空比20%的100mV/mm脉冲直流电场条件下,分别记录两种培养方法生长锥在2小时内不同时间的位点,计算其与原点连线与预计生长方向(即电力线垂直线)之间的角度,分别计算平均值。结果:在Laminin铺片的培养体系中,100mV/mm的稳态直流电场和占空比20%的脉冲直流电场均可引起神经突向负极方向偏转;而在PDL铺片的培养体系中,神经突向正极方向偏转。在无电场的培养体系中,神经突无明显偏转。结论:稳态和脉冲直流电场对体外培养螺旋神经元的神经突生长有导向作用;导向的方向受培养底物的影响。第三部分电荷平衡式双相脉冲电场对体外培养螺旋神经元神经突生长方向的影响目的:观察电荷平衡式双相脉冲电场对体外培养螺旋神经元神经突生长方向的影响。方法:采用Laminin包被玻片,用组织块法体外培养螺旋神经元。选择有“自由神经突”形成的玻片移植到结合环状电极电场的活细胞观察系统中,使神经突末端的主干与两电极中心连线平行,并使生长锥位于经过其中一个电极中心的连线垂直线上,朝向电极方向。电极间距2mm,刺激频率250 Hz,振幅±1mA,相宽100us,相间断路间隔20us和短路相3.78ms,分别选取3个位点作为研究对象,使其与电极距离分别为两电极间距的1/2、1倍和2倍,即1mm,2mm和4mm处。记录4小时内生长锥的位移轨迹。并以生长锥位于1倍极间距处不施加电场作为对照。结果:电场作用下,位于1/2、1倍和2倍极间距处的生长锥时均向远离电极侧偏转。无电场作用时,神经突无明显偏转倾向。结论:电荷平衡式双相脉冲电场中,体外培养螺旋神经元神经突有远离电极的倾向。在远离电极时,这种倾向逐渐变小。更多还原

【Abstract】 PartⅠStudy on the construction of neurite guidance system of spiral ganglion neurons by electric fields in vitroObjectives: To establish a neurite guidance system of spiral ganglion neurons by electric fields in ivtro.Methods: The spiral ganglion of postnatal day 4-6 Sprague-Dawley rats were dissected out. Spiral ganglion neurons were cultured by dissociated culture method or explants culture method. The coating materials included Laminin, PDL, PDL plus Laminin, Collagen I and none. The antibody of Neurofilament, MAP2 and Tau-1 were used to identify the neurite of spiral ganglion neurons. The systems of electric fields were constructed by inducing Pt-Ir wires, rings or electrode array, which were differently connected with steady DC power, Function/Arbitrary waveform generator or Cochlear Implant signal processor. The traces of growth cones were recorded by taking photos just before and after the electric stimulus in a CO₂ incubator or by time lapse module in live cell imaging （LCI） system after transplanting culture.Results: It was much easier for the neurites of spiral ganglion neurons to become "free" neurites without interference by surrounding cells in explants culture system than that in dissociated culture system. More neurites were found on slices coating with Laminin, PDL or PDL plus Laminin than on ones coating with Collagen I and none. The neurites could be labeled by the antibody of Neurofilament and Tau-1, but not by the antibody of MAP2. The method of time lapse recording in LCI system after transplanting culture was helpful to maintain a steady environment for neurite extending. Furthermore, it made the orientation of neurites in electric fields much easy, which is essential to the investigation on neurite guidance and helpful to improve investigation efficiency.Conclusions: Explants culture on slices coating with Laminin, PDL or PDL plus Laminin is suitable for neurite guidance investigation on spiral ganglion neurons. Transplanting culture and time lapse recording in LCI system is helpful to an efficient investigation on neurite guidance of spiral ganglion neurons by electric fields. PartⅡThe effect of steady and pulsed electric fields on the neurite growth direction of spiral ganglion neurons in vitroObjectives: To investigate the effect of steady and pulsed electric fields on neurite growth direction of spiral ganglion neurons in vitro.Methods: The spiral ganglion neurons were cultured on slices coated with Laminin or PDL by explants culture. The slices with "free neurite" were transplanted into LCI system with the direction of neurite terminals perpendicular to the vector in parallel-electrodes electric fields. The locations of growth cones in different time were recorded by taking photos. The turning angles were measured in different culture systems with no electric fields, steady or pulsed 100mV/mm DC electric fields.Results: The neurites cultured on Laminin coated slices turned to the cathode by steady and pulsed 100mV/mm DC electric fields, while the neurites cultured on PDL coated slices turned to the anode. The neurites extended almost straightly in culture systems without electric field.Conclusions: Steady and pulsed DC electric fields could both steer the direction of neurite extension of spiral ganglion neurons. Furthermore, the directions were different on slices coated with different materials.PartⅢThe effect of charged-balanced biphasic pulsed electric fields on the neurite growth direction of spiral ganglion neurons in vitroObjectives: To investigate the effect of charged-balanced biphasic pulsed electric fields on the neurite growth direction of spiral ganglion neurons in vitro.Methods: The spiral ganglion neurons were cultured on slices coated with Laminin by explants culture. The slices with "free neurite" were transplanted into LCI system with the direction of neurite terminals parallel to the line of two ring electrodes. The distance between the two electrodes （D） was 2 mm. The growth cones were oriented on the location 1 mm （1/2D）, 2 mm （D） and 4 mm （2D） away to one of the electrodes. And the growth cones 2 mm （D） away to one of the electrodes in culture system without electric fields were also investigated as a contrast. The traces of growth cones were recorded by taking photos, which followed by turning angles measurement.Results: The neurites 1 mm （1/2D）, 2 mm （D） and 4 mm （2D） away to the electrodes turned off the electrodes, while the directions of neurites in culture systems without electric field had no obvious tendency.Conclusions: The neurites of spiral ganglion neurons in charged-balanced biphasic pulsed electric fields had a tendency to turn away from the electrodes in vitro.更多还原