高通量筛选酸敏感离子通道1a抑制剂研究  

High Thoughtout Screening of ASIC1a Inhibitors

作　　者：羿菲[1] 李小曼[1] 白宁[1] 孙威[1] 姜波[1] 张莹[1] 宋晓宇[1] 

机构地区：[1]中国医科大学转化医学研究院,辽宁省沈阳市110122

出　　处：《中国全科医学》2016年第17期2033-2037,共5页Chinese General Practice

基　　金：国家自然科学基金资助项目(81502414);教育部"创新团队发展计划"资助项目(IRT13101)

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Ca^(2+)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Ca^(2+)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,Ca^(2+)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。Objective To establish the FRT cell of Fisher rats of stable in vitro ASIC1a overexpression,Ca2 ＋ -sensitive fluorescent dye Fura-2 / AM and cytotoxicity test（LDH releasing method）were used to determine the activity of ASIC1a, to discuss the feasibility of high throughput screening of ASIC1a inhibitors. Methods From January 2014 to February 2016, molecular biology method was used to establish ASIC1a overexpression vector pcDNA3. 1 / myc-His-mASIC1a. By cell transfection technique,we established FRT cell of Fisher rats with stable ASIC1a overexpression. The cells were divided into two groups：control group（FRT group）and experiment group（FRT cells with stable ASIC1a 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 pH 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 pH 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 pH values（ P 〈 0. 05）. Conclusion Stable ASIC1a 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 ASIC1a, which make the high thoughtout screening of AS

关 键 词：高通量筛选分析 酸敏感离子通道1a 上皮细胞 乳酸脱氢酶 

分 类 号：R965.1[医药卫生—药理学]