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酸刺激对大鼠颈动脉体I 型细胞酸敏感离子通道亚型表达的影响

李丹, 陈海锋, 唐玉红, 周华, 徐建国, 陈丽, 郑煜

四川大学 华西基础医学与法医学院生理学教研室，成都610041；华西医院神经外科，成都610041

摘要

本研究在原代培养大鼠颈动脉体(carotid body, CB) I 型细胞中检测酸敏感离子通道(acid-sensing ion channels, ASICs) 亚型ASIC1a 和ASIC3 的表达情况，并观察酸刺激对其表达的影响。实验取体重50~100 g Sprague-Dawley 大鼠双侧CB 进 行原代培养，采用免疫荧光技术检测培养细胞有无CB I 型细胞特征性标记物酪氨酸羟化酶(tyrosine hydroxylase, TH)的表 达，以鉴定培养细胞的类型。采用免疫荧光双标技术，分别检测ASIC1a 和ASIC3 在TH 阳性细胞中的表达情况。以两种 酸性培养基(pH 值分别为6.8 和6.2)作用CB 细胞24 h，pH7.3 培养基为对照组，采用半定量RT-PCR 技术检测ASIC1a 和 ASIC3 mRNA 表达的变化。免疫荧光鉴定显示，原代培养的细胞93% 以上是TH 表达阳性细胞。免疫荧光双标显示，TH 阳性细胞胞浆同时表达ASIC1a 或ASIC3。RT-PCR 结果显示，ASIC1a mRNA 水平在不同酸刺激下变化不明显；ASIC3 mRNA 水平在pH6.8 组变化不明显，而在pH6.2 组显著低于pH7.3 对照组和pH6.8 组。上述结果提示，在转录水平上， 酸刺激对CB I 型细胞不同亚型酸敏感离子通道的作用是不同的，对ASIC3 具有下调作用，而对ASIC1a 没有明显的调控作用。

关键词： 颈动脉体; 原代细胞培养; 酸敏感离子通道

分类号：R332 ；Q78 6

[Influence of acid stimulation on expression of acid-sensing ion channel 1a and 3 in type I cells of rat carotid body.] [Ariticle in Chinese]

LI Dan, CHEN Hai-Feng, TANG Yu-Hong, ZHOU Hua, XU Jian-Guo, CHEN Li, ZHENG Yu

Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China； Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu 610041, China

Abstract

The experiments were carried out to test whether acid-sensing ion channel 1a and 3 (ASIC1a and ASIC3) were expressed on the primarily cultured type I cells of rat carotid bodies (CBs) and whether the expression of the channels was affected by acid stimulation. The Sprague-Dawley rats of either sex (50–100 g) were used. The CBs were isolated and primarily cultured. The immunofluorescent technique was used to detect the expression of tyrosine hydroxylase (TH), a specific marker of type I cells, in order to identify the type of the cultured cells. The double-label immunofluorescent technique was used to dectect the expression of ASIC1a and ASIC3 on the TH-positive type I cells. To detect the influence of acid stimulation on the expressions of ASIC1a and ASIC3, each batch of primarily cultured cells were randomly divided into pH7.3 group (control group), pH6.8 group and pH6.2 group (n=9 in each group). The cells in above three groups were treated with pH7.3, pH6.8 and pH6.2 mediums for 24 h, respectively, and then the mRNA expressions of ASIC1a and ASIC3 in type I cells were detected by semi-quantitative RT-PCR technique. The results showed that more than 93% of the primarily cultured CB cells were TH-positive, indicating that most of the cultured cells were type I cells. Furthermore, all TH-positive cells expressed ASIC1a or ASIC3. After the cells were treated with acid stimulation, the amount of ASIC1a mRNA did not change significantly (P>0.05 vs control group); the amount of ASIC3 mRNA had no significant change in pH6.8 group compared with that in control group, but decreased significantly in pH6.2 group (P<0.01 vs control group, P<0.05 vs pH6.8 group). It is concluded that acid stimulation down-regulates the level of ASIC3 mRNA, but has no effect on the level of ASIC1a mRNA.

Key words: Carotid body; primary cell culture; acid-sensing ion channels

收稿日期：2008-07-15　　录用日期：2008-10-28

通讯作者：郑煜　　E-mail: yzheng@scu.edu.cn

引用本文：

李丹, 陈海锋, 唐玉红, 周华, 徐建国, 陈丽, 郑煜. 酸刺激对大鼠颈动脉体I 型细胞酸敏感离子通道亚型表达的影响[J]. 生理学报 2009; 61 (1): 43-48.

LI Dan, CHEN Hai-Feng, TANG Yu-Hong, ZHOU Hua, XU Jian-Guo, CHEN Li, ZHENG Yu. [Influence of acid stimulation on expression of acid-sensing ion channel 1a and 3 in type I cells of rat carotid body.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 43-48 (in Chinese with English abstract).