上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2017, Vol. 37 ›› Issue (11): 1459-.doi: 10.3969/j.issn.1674-8115.2017.11.002

• 论著（基础研究） • 上一篇    下一篇

胆红素导致 SD 大鼠脑干神经元突触小体 Ca2+ 过载

李丹萍，赖轲，王际平，时海波   

  1. 上海交通大学附属第六人民医院耳鼻咽喉头颈外科，上海 200233
  • 出版日期:2017-11-28 发布日期:2018-01-10
  • 通讯作者: 时海波，电子信箱：haibo99@hotmail.com
  • 作者简介:李丹萍（1989—），女，住院医师，硕士生；电子信箱：danping_710@163.com
  • 基金资助:
    国家自然科学基金面上项目（81371092，81570909）；上海市教育委员会高峰高原学科建设计划（20152233），上海申康医院发展中心促进市级 医院临床技能与临床创新能力三年行动计划（16CR3041A）

Bilirubin-induced calcium overload in synaptosomes isolated from brainstem neurons of rats#br#

LI Dan-ping, LAI Ke, WANG Ji-ping, SHI Hai-bo   

  1. Department of Otorhinolaryngology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China
  • Online:2017-11-28 Published:2018-01-10
  • Supported by:
     National Natural Science Foundation of China, 81371092，81570909; Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support, 20152233; Clinical Research Promotion Plan of Shanghai Shen Kang Hospital Development Center, 16CR3041A

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摘要： 目的 · 探讨胆红素暴露对 SD 大鼠脑干神经元突触小体游离钙离子浓度（[Ca2+]i）的影响。方法 · 采用出生后 7 ～ 14 d 的 SD 大鼠 40 只，随机分成 3 组，分别为对照组、胆红素组 ( 浓度分别为 0.1、1、10 μmol/L）以及胆红素 + 甘氨酸熊脱氧胆酸（GUDCA） 干预组。用蔗糖密度梯度离心法提纯脑干神经元突触小体；采用光学显微镜及透射电子显微镜验证突触小体活性，以 OG-BAPTA 作 为钙离子荧光探针，共聚焦激光扫描显微镜下观察不同条件下突触小体荧光强度的实时变化。结果 · 获得了具有活性的SD 大鼠脑 干神经元突触小体。在对照组中随着观察时间的推移，突触小体荧光强度缓慢升高；胆红素组表现更为强烈、与暴露时间及浓度 相关的升高（主体内、主体间效应检验，P<0.05）；单纯给予GUDCA 并未改变实验体系中的荧光强度升高趋势（与对照组比较， P=0.656），但 GUDCA 干预降低了胆红素暴露所致荧光强度的升高程度，尤其是较高浓度（10 μmol/L）胆红素，差异具有统计学意义 （P=0.000）。 结论 · 胆红素能使突触小体发生钙超载，并与暴露浓度、时间呈正相关；GUDCA 对胆红素所致的突触小体钙超载具有抑 制作用，可能通过该途径拮抗胆红素神经毒性。

关键词: 胆红素, 突触小体, 游离钙离子, 甘氨酸熊脱氧胆酸, 免疫荧光探针

Abstract:

Objective · To observe real-time changes of calcium concentration ([Ca2+]i) exposure to bilirubin in synaptosomes isolated from brainstem nucleus of rats.  Methods · Forty P7-14 SD rats were randomly assigned to three groups: control group, bilirubin group (with levels of 0.1, 1 and 10 μmol/L) and bulirubin plus glycoursodeoxycholic acid (GUDCA) group. The synaptosomes were purified from brainstem nucleus by sucrose density gradient centrifugation. After loading OG-BAPTA in synaptosomes, two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy.  Results · Synaptosomes with well biological activity were obtained from brainstem of the SD rats. In the control group, a progressive increase in fluorescent intensity of [Ca2+]i was detected. In the bilirubin group, acuter increases in fluorescent intensity were observed in all levels of bilirubin, with a manner of both concentration and time-dependent (P<0.05). Fluorescent intensity of [Ca2+]i was reduced  in the present of GUDCA, which was not significant compared with the control group (P=0.656). However, GUDCA could abate the increase of fluorescent intensity of [Ca2+]i induced by bilirubin exposure, of which showing significant decrease in 10 μmol/L bilirubin exposure (P=0.000).  Conclusion · Bilirubin could induce calcium overload in synaptosomes. GUDCA could abate bilirubin-induced calcium overload in synaptosomes, possibly explaining its protection effect of neurons from bilirubin neurotoxicity.

Key words:  bilirubin, synaptosome, intracellular calcium, glycoursodeoxycholic acid, fluorescent probe