一种基于高内涵成像分析技术的快速检测RhoA蛋白激活的方法  

A rapid detection method for activated RhoA proteins based on high-content image anylysis

作　　者：周亚男 曲颖 王劭文 孙毅 苏瑞斌 ZHOU Yanan;QU Ying;WANG Shaowen;SUN Yi;SU Ruibin(Nanjing University of Chinese Medicine,Beijing Key Laboratory of Neuropsychopharmacology,Nanjing 210023,China;State Key Laboratory of National Security Specially Needed Medicines,Beijing Key Laboratory of Neuropsychopharmacology,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]南京中医药大学,江苏南京210023 [2]军事医学研究院国家安全特需药品全国重点实验室,神经精神药理学北京市重点实验室,北京100850

出　　处：《中国药理学与毒理学杂志》2024年第11期839-845,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：全国重点实验室自主研究课题重点项目(LTMC2022ZZ003)。

摘　　要：目的 利用高内涵成像分析系统(HCIS)建立快速检测Ras同源基因家族成员A(RhoA)蛋白激活的方法。方法 将人胚胎肾细胞293(Hek293)或仓鼠卵巢细胞(CHO)接种在96孔板中,待细胞贴壁后进行饥饿处理。Hek293细胞分别使用浓度为0(溶剂对照组),10^(-11),10^(-10),10^(-9),10^(-8),10^(-7),10^(-6),10^(-5)和10^(-4)mol·L^(-1)RhoA激动剂诺考达唑孵育3,10和30 min;CHO细胞分别使用相同浓度诺考达唑、溶血磷脂酸(LPA)和钙肽素孵育3,10和30 min。孵育结束用3.7%甲醛溶液进行固定后,使用烟酸己可碱(hoechst)和罗丹明标记鬼笔环肽,室温避光染色,利用HCIS拍照并对图像进行分析和统计,以平均荧光强度(MFI)的变化评估药物对RhoA蛋白的激活。结果 与溶剂对照组相比,处理Hek293细胞3 min后诺考达唑10^(-10)~10^(-6)mol·L^(-1)组MFI显著升高(P<0.01),处理30 min后10^(-10)~10^(-4)mol·L^(-1)组MFI升高(P<0.01),表明RhoA蛋白被激活。在CHO细胞上,与溶剂对照组相比,诺考达唑10^(-10)~10^(-6)mol·L^(-1)处理10和30 min MFI均升高(P<0.05,P<0.01);LPA 10^(-11)~10^(-4)mol·L^(-1)处理3 min和10^(-11),10^(-8)~10^(-4)mol·L^(-1)处理10 min及10^(-11)~10^(-9),10^(-7),10^(-6),10^(-4)mol·L^(-1)处理30 min MFI均升高(P<0.05,P<0.01);钙肽素10^(-11)~10^(-6),10^(-4)mol·L^(-1)处理10 min及10^(-11)和10^(-4)mol·L^(-1)处理30 min MFI均升高(P<0.05,P<0.01),均表明RhoA蛋白被有效激活。结论 成功建立了快速检测RhoA蛋白激活的方法,可实现高通量、快速、高效、便捷地对RhoA蛋白的激活进行检测。OBJECTIVE To develop a rapid method for detection of activated RhoA protein using the high content imaging system(HCIS).METHODS Hek293 or CHO cells were seeded in 96-well plates and subjected to starvation treatment after attachment.Hek293 cells were incubated with nocodazole,a RhoA agonist,at concentrations of 0(vehicle control),10^(-11),10^(-10),10^(-9),10^(-8),10-7,10^(-6),10^(-5) and 10^(-4) mol·L^(-1) for 3,10 and 30 min respectively.CHO cells were incubated with nocodazole,lyso-phosphatidic acid(LPA)and calpain at the same concentrations for 3,10 and 30 min respectively.Imme-diately after incubation,the cells were fixed with 3.7%formaldehyde solution and stained using Hoechst and rhodamin phallodin at room temperature and protected from light.Images were captured using HCIS and analyzed statistically.Changes in the mean fluorescence intensity(MFI)were used to assess the activation of RhoA protein by the drugs.RESULTS Compared with the vehicle control group,the MFI of Hek293 cells treated with nocodazole for 3 min significantly increased at concentra-tions ranging from 10^(-10) to 10^(-6) mol·L^(-1)(P<0.01).When the treatment duration was extended to 30 min,MFI elevations were observed at concentrations between 10^(-10) and 10^(-4) mol·L^(-1)(P<0.01),indicating the activation of RhoA protein.In CHO cells,compared with the vehicle control group,MFI was increased after 10^(-10)-10^(-6) mol·L^(-1) nocodazole treatment of 10 min and 30 min(P<0.05,P<0.01).Similarly,MFI was also increased under various conditions of LPA and calpeptin treatment.LPA 10^(-11)-10^(-4) mol·L^(-1) treatment of 3 min and 10^(-11),10^(-8)-10^(-4) mol·L^(-1) treatment of 10 min and 10^(-11)-10^(-9),10-7,10^(-6),10^(-4) mol·L^(-1) treatment of 30 min all resulted in an elevated MFI(P<0.05,P<0.01).Calpeptin 10^(-11)-10^(-6),10^(-4) mol·L^(-1) treatment of 10 min and 10^(-11) and 10^(-4) mol·L^(-1) treatment of 30 min also resulted in an elevated MFI(P<0.05,P<0.01).These results indicated that RhoA protein was effectively activated.C

关 键 词：Ras同源基因家族成员A 蛋白检测 高内涵成像分析 荧光染色 高通量 

分 类 号：R965[医药卫生—药理学]