【摘要】 目的建立体外稳定过表达酸敏感离子通道1a（ASIC1a）的Fisher大鼠甲状腺上皮细胞（FRT细胞）,采用Ca²⁺敏感荧光染料Fura-2/AM和细胞毒性检测〔乳酸脱氢酶（LDH）释放法〕测定ASIC1a活性,探讨高通量筛选ASIC1a抑制剂的可行性。方法2014年1月—2016年2月,利用分子生物学方法,构建ASIC1a过表达载体pc DNA3.1/myc-His-m ASIC1a,通过细胞转染技术建立了稳定过表达ASIC1a的Fisher大鼠FRT细胞。将细胞分成两组,对照组（FRT细胞）和实验组（稳定过表达ASIC1a的FRT细胞）,并分别未负载和负载Ca²⁺敏感荧光染料Fura-2/AM,采用酶标检测仪测定双波长（340 nm和380 nm）的值,计算F340/F380。对照组和实验组细胞经不同p H值酸液（p H值7.5、7.0、6.5、6.0、5.5）刺激后,采用酶标检测仪测定450 nm波长处LDH释放量。结果对照组与实验组未负载Fura-2/AM细胞F340/F380比较,差异无统计学意义（P>0.05）;实验组负载Fura-2/AM细胞F340/F380较对照组升高（P<0.05）;对照组和实验组负载Fura-2/AM细胞F340/F380较未负载Fura-2/AM细胞升高（P<0.05）。p H值6.5、6.0、5.5时,实验组细胞LDH释放量大于对照组（P<0.05）;对照组和实验组细胞不同p H值时LDH释放量比较,差异有统计学意义（P<0.05）。结论本研究成功建立稳定过表达ASIC1a的FRT细胞,Ca²⁺敏感荧光染料Fura-2/AM和细胞毒性检测（LDH释放法）两种方法测定ASIC1a活性,使高通量筛选ASIC1a抑制剂成为可能,为发现高选择性和高活性的天然小分子ASIC1a抑制剂奠定了基础。更多还原

【Abstract】 Objective To establish the FRT cell of Fisher rats of stable in vitro ASIC1 a overexpression,Ca²⁺-sensitive fluorescent dye Fura-2/AM and cytotoxicity test（ LDH releasing method） were used to determine the activity of ASIC1 a,to discuss the feasibility of high throughput screening of ASIC1 a inhibitors.Methods From January 2014 to February 2016,molecular biology method was used to establish ASIC1 a overexpression vector pc DNA3.1/myc-His-m ASIC1 a.By cell transfection technique,we established FRT cell of Fisher rats with stable ASIC1 a overexpression.The cells were divided into two groups:control group（ FRT group） and experiment group（ FRT cells with stable ASIC1 a overexpression）;two groups were loaded or not loaded with Ca²⁺-sensitive fluorescent dye Fura-2/AM,and enzyme mark detector was employed to determine dual-wave length（ 340 nm and 380 nm） and the ratio of F340/F380.After the cells in control group and experiment group were stimulated by acid liquid of different p H values（ 7.5,7.0,6.5,6.0 and 5.5）,the LDH release at 450 nm wave length was determined using enzyme mark detector.Results Control group and experiment group were not significantly different in F340/F380 of cells loaded with Fura-2/AM（ P > 0.05）;experiment group was higher than control group in F340/F380 of cells loaded with Fura-2/AM（ P < 0.05）;control group and experiment group had higher F340/F380 in cells loaded with Fura-2/AM than in cells not loaded with Fura-2/AM（ P < 0.05）.When p H value were 6.5,6.0 and 5.5,experiment group was higher than control group in LDH release（ P < 0.05）;control group and experiment group were significantly different in LDH release in the cases of different p H values（ P < 0.05）.Conclusion Stable ASIC1 a overexpression cells are established,and Ca²⁺-sensitive fluorescent dye Fura-2/AM and cytotoxicity test（ LDH releasing method） are used to determine the activity of ASIC1 a,which make the high thoughtout screening of ASIC1 a inhibitors possible and lay a foundation for finding natural small molecule ASIC1 a inhibitors with specificity and high activity.更多还原