中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 285-288.doi: 10.3969/j.issn.1673-8225.2010.02.022

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

双分离法记录Wistar大鼠神经元的全细胞电流

沈佩同1，毕  平1，李  刚2   

  1. 1天津医科大学生物医学工程系，天津市  300070；2天津大学精密仪器与光电子学院，天津市  300072
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 毕 平，副教授，硕士生导师，天津医科大学生物医学工程系，天津市 300070 Biping63@yahoo.com.cn
  • 作者简介:沈佩同★，男，1985年生，河南省西峡县人，汉族，天津医科大学生物医学工程系在读硕士，主要从事物理因子对脑皮质以及海马细胞离子通道的研究。

Dual-dissociation method to record whole-cell current of wistar rat neurons 

Shen Pei-tong1, Bi Ping1,Li Gang2   

  1. 1 Department of Biomedical Engineering, Tianjin Medical University, Tianjin  300070, China; 2 College of Precision Instrument & Opto-electronic Engineering, Tianjin University, Tianjin  300072, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Bi Ping, Associate professor, Master’s supervisor, Department of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China
  • About author:Shen Pei-tong★, Studying for master’s degree, Department of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China

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1

摘要：

背景：细胞培养与通道电流记录是全细胞膜片钳实验的主要难点。
目的：介绍一种简单可行的降低全细胞膜片钳实验方法，将细胞急性分离与电流的分离技术结合起来，以提高工作效率，缩短实验的时间，从根本上降低膜片钳实验的难度。
方法：SPF级出生4~7 d的wistar大鼠40只，雌雄不限。采用改良的急性分离的方法制备Wistar大鼠脑皮质细胞，将大鼠脑皮质切成400~600 μm厚度的薄片，在人工脑脊液中通混合气静止1 h，并通以氧气。将脑组织块放入含有16 u/mL( type X )和2 u/mL(type XIV) 蛋白水解酶的人工脑脊液中，孵育60 min，清除消化酶。在全细胞电压钳制模式下，保持电位-80 mV，给予-60 mV到60 mV的去极化脉冲刺激，步阶为+10 mV，刺激波宽160 ms。记录到跨膜总电流，在全细胞电极液里面加入70 mmol/LCsCl，70 mmol/L CsF；在外液中先后加入11 μmol/L  阻断剂河豚毒素、30 mmol/L的氯化四乙胺、1 mmol/L的4-AP。分别记录内向钠电流，瞬时外向钾电流和延迟整流钾电流，结果用clampfit分析处理。主要观察：①细胞的形态学观察。②全细胞电流的记录。③内向钠电流的记录。④外向钾电流的记录。
结果与结论：细胞空间立体结构强，表面光滑，有完整的树突或者轴突，且细胞的活性可以在25 ℃室温下维持8~10 h。在外液中加入1 μmol/L的河豚毒素基本上可以阻断钠电流；30 mmol/L的氯化四乙胺和1 mmol/L的4-AP可以阻断外向钾电流。结果表明，改良的细胞急性分离方法细胞功能完好。通过电流分离技术，不改变细胞外液和电极液，仅需添加特异阻断剂，可记录到内向钠电流，瞬时外向钾电流以及延迟整流钾电流，较之传统方法可显著提高工作效率。 

关键词: 急性分离法, 电流分离法, 全细胞电流, 膜片钳

Abstract:

BACKGROUND: The main difficulties of whole-cell patch clamp are cell culture and whole-cell current recording.
OBJECTIVE: To reduce the difficulty of patch-clamp experiment by combining acute cell isolation with current separation technique.
METHODS: A total of 40 wistar rats aged 4-7 days, irrespective of genders, were selected. The pallium of wistar rats were cut into slices with 400-600 μm, rested for 1 hour in artificial cerebrospinal fluid with gas mixture, simultaneously put in oxygen. The brain tissues were placed into artificial cerebrospinal fluid containing
16 u/mL( type X ) and 2 u/mL(type XIV) proteinase, incubated for 60 minutes, and cleared the digestive enzyme. Under the whole-cell voltage clamp mode, the potential was hold at -80mV, depolarizing pulse stimulation from -60 mV to 60 mV, 10 mV step and 160 ms width. The total Tran membranes current was recorded, and then 70 mmol/LCsCl, 70mmol/L CsF was 11 μmol/L Na+ channel blockers tetrodotoxin, 30 mmol/L tetraethylammonium, 1 mmol/L 4-AP was successively added into extracellular fluid. The inward sodium current, transient outward potassium current and delayed rectifier potassium current was recorded, and then the result was analyzed using clampfit. 
MAIN OUTCOME MEASURES: ①Cell morphological observation; ②Whole-cell current recording; ③Inward sodium current recording; ④Outward potassium current recording.
RESULTS AND CONCLUSION: The cell had clearly three-dimensional structure, smooth surface and whole neurodendrite or neuraxon. The cell viability could maintained for 8-10 hours at 25 ℃ temperature. The added 11 tetrodotoxin in extracellular fluid could block sodium current. The outward potassium current could be blocked by tetraethylammonium with 30 mmol/L and 4-AP with 1 mmol/L. The cells harvested by modified rapid dissociation have good functions. Using current separation method, only specific blockers are needed, without extracellular fluid or electrode solution replacing, which can record the inward sodium current, transient outward potassium current, as well as delayed rectifier potassium current. This method can obviously improve the work efficiency than traditional one.

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