摘要
目的:构建基于钙激活氯离子通道(CaCC)的高通量P2Y2嘌呤受体(P2ry2)调节剂细胞筛选模型。方法:采用RT-PCR方法验证Fischer大鼠甲状腺滤泡上皮细胞(FRT细胞)中P2ry2的mRNA表达水平,切胶回收PCR扩增产物并进行序列测序和比对,进一步应用Western blot检测FRT细胞的P2ry2蛋白表达水平。构建钙激活氯离子通道ANO1和对卤族元素敏感的黄色荧光蛋白双突变体YFP-H148Q/I152L真核表达载体,应用脂质体转染、抗生素筛选和有限稀释,获取共表达ANO1和YFP-H148Q/I152L的FRT细胞。采用倒置荧光显微镜观察共表达ANO1和YFP-H148Q/I152L细胞的情况,并应用荧光淬灭动力学实验验证模型的有效性;荧光淬灭动力学实验验证细胞模型是否可筛选P2ry2调节剂,并应用Fura-2/AM荧光探针法检测细胞内游离钙的变化,探究胞内Ca2+浓度与P2ry2调节剂的剂量依赖关系;用Z'因子评价本模型的稳定性和可重复性。结果:FRT细胞内源性表达P2ry2;倒置荧光显微镜中观察到ANO1在细胞膜上表达绿色荧光,YFP-H148Q/I152L在胞浆中表达绿色荧光,成功构建了共表达ANO1和YFP-H148Q/I152L的细胞模型;荧光淬灭动力学实验证实该细胞模型可筛选P2ry2调节剂,应用Fura-2表明该细胞模型可敏感地检测胞浆内钙离子浓度的变化且Ca2+浓度与P2ry2调节剂及相对荧光强度呈剂量依赖关系;Z'因子为0.75,说明该细胞模型可以高通量筛选P2ry2的调节剂且有良好的稳定性与可重复性。结论:成功构建了一种经济简便快捷高效的P2ry2调节剂细胞筛选模型,适用于P2ry2调节剂的发现与评价。
AIM:To construct a high-throughput screening cell model for P2Y2 purinergic receptor(P2ry2)modulators based on calcium-activated chloride channel(CaCC).METHODS:The mRNA expression of P2ry2 in Fischer rat thyroid(FRT)cells was detected by RT-PCR,and the PCR products were collected and sequenced.The pro⁃tein expression of P2ry2 in the FRT cells was also detected by Western blot.The eukaryotic expression vectors ANO1 and YFP-H148Q/I152L were constructed.The FRT cells co-expressing ANO1 and YFP-H148Q/I152L were obtained by lipo⁃some transfection,antibiotic screening and limited dilution.The expression of ANO1 and YFP-H148Q/I152L in the cells was observed under fluorescent inverted microscope.The validation of the cell model for screening P2ry2 modulators was verified by the fluorescence quenching kinetics tests,and intracellular free calcium was analyzed by Fura-2 staining to in⁃vestigate the dose-dependent relationship between intracellular calcium concentration and P2ry2 modulators.Z'factor was applied to evaluate the stability and repeatability of the cell model.RESULTS:P2ry2 were endogenously expressed in the FRT cells.The expression of ANO1 on the cell membrane and the expression of YFP-H148Q/I152L in the cytoplasm were observed under fluorescent inverted microscope.The cell model was successfully constructed.The fluorescence quenching kinetics test confirmed the cell model for screening P2ry2 modulators was constructed successfully,and the calcium con⁃centration in cytoplasm was increased rapidly after the addition of a small amount of P2ry2 agonist,indicating that the cell model was sensitive for detecting the calcium concentration in cytoplasm.The calcium concentration in cytoplasm,P2ry2 modulators and the slope of fluorescence change were in a dose-dependent manner,respectively.The Z'factor was 0.75,indicating that the established cell model was able to use for high-throughput screening of P2ry2 modulators with excellent stability and repeatability.CONCLUSION:A simple,economical,and efficient cell screening model of P2ry2 modula⁃tors is successfully constructed,which is suitable for the detection and evaluation of P2ry2 modulators.
作者
郭佳琦
肖云萍
丁旭
解宇浩
张嘉琪
郝峰
GUO Jia-qi;XIAO Yun-ping;DING Xu;XIE Yu-hao;ZHANG Jia-qi;HAO Feng(College of Laboratory Medicine,Jilin Medical College,Beihua University,Jilin 132013,China.;School of Laboratory Medicine,Beihua University,Jilin 132013,China.)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2020年第11期2105-2112,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金项目资助(No.81601234)
吉林省教育厅基金资助课题(No.JJKH20170418KJ,No.JJKH20191056KJ)
吉林医药学院启动资金(No.2017KYQD001)
吉林省卫生与健康技术创新项目(No.2018J113)
国家级和吉林省大学生创新创业训练计划(No.201713743004,No.201913706071)。
