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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50～100g 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CBI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24h,pH7.3培养基为对照组,采用半定量RT-PCR技术检测ASIC1a和ASIC3mRNA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;ASIC3mRNA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CBI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。
In this study, we detected the expression of ASIC1a and ASIC3 in acid-sensing ion channels (ASICs) in primary cultured rat carotid body (CB) type I cells and observed the effect of acid stimulation on their expression Impact. Experiments Bilateral CB of 50 ~ 100g Sprague-Dawley rats were cultured in primary culture, the expression of tyrosine hydroxylase (TH), a characteristic marker of CBI cells, was detected by immunofluorescence technique, To identify the type of cultured cells. Immunofluorescence double labeling was used to detect the expression of ASIC1a and ASIC3 in TH-positive cells. CB cells were treated with two acidic media (pH values of 6.8 and 6.2, respectively) at 6.2 and pH7.3 medium as control group. Semi-quantitative RT-PCR was used to detect the changes of ASIC1a and ASIC3 mRNA expression. Immunofluorescence assay showed that more than 93% of primary cultured cells were TH-positive cells. Double immunofluorescence staining showed that TH-positive cytoplasm expressed ASIC1a or ASIC3 simultaneously. RT-PCR results showed that ASIC1a mRNA level did not change significantly under different acid stimulation; ASIC3 mRNA level did not change significantly in pH6.8 group, but was lower than pH7.3 group and pH6.8 group in pH6.2 group. The above results suggest that acid stimulation at the transcriptional level has different effects on the acid-sensitive ion channels of different subtypes of CBI-type cells, downregulation of ASIC3, and no significant regulation of ASIC1a.