α_(1A)-肾上腺素能受体与增强型绿色荧光蛋白标记的活化T细胞核因子2稳定共表达细胞的构建

Establishment of cells stably co-expressing with α_(1A)-adrenergic receptor and enhanced green fluorescent protein tagged nuclear factor of activated T cells 2

目的建立α_(1A)-肾上腺素能受体(α_(1A)-AR)与增强型绿色荧光蛋白(EGFP)标记的活化T细胞核因子2(NFAT2)稳定共表达细胞。方法①将pcDNA3.1-α_(1A)-AR-3×FLAG重组质粒转染至U2OS-EGFPNFAT2细胞,经潮霉素B(Hygro-B)200 mg·L^(-1)压力筛选后加入α_(1A)-AR激动剂去甲肾上腺素(NE,10μmol·L^(-1))孵育30 min,通过高内涵筛选系统检测细胞核内绿色荧光强度,验证EGFP-NFAT2核转位,筛选得到稳定表达α_(1A)-AR的U2OS-EGFP-NFAT2-α_(1A)-AR细胞。②采用实时荧光定量PCR(RT-qPCR)和Western印迹法检测该细胞和对照细胞U2OS-EGFP-NFAT2中α_(1A)-AR mRNA和蛋白的表达水平。③将U2OS-EGFP-NFAT2-α_(1A)-AR细胞接种于96孔板,分别加入NE(10^(-8)~10^(-5) mol·L^(-1))或α2-AR激动剂右美托咪定(DMED,10^^(-8.8)~10^(-5) mol·L^(-1))孵育30 min,通过高内涵筛选系统检测EGFP-NFAT2核转位。④将U2OS-EGFP-NFAT2-α_(1A)-AR细胞分为溶剂对照组、α1-AR拮抗剂萘派地尔(1μmol·L^(-1))组、NE(1μmol·L^(-1))组、萘派地尔+NE(各1μmol·L^(-1)共孵育)组、α2-AR拮抗剂阿替美唑(0.1μmol·L^(-1))组、DMED(0.1μmol·L^(-1))组、阿替美唑+DMED(各0.1μmol·L^(-1)共孵育)组和萘派地尔+DMED(萘派地尔1μmol·L^(-1)与DMED 0.1μmol·L^(-1)共孵育)组,药物孵育时间均为30 min,通过高内涵筛选系统检测EGFP-NFAT2核转位,验证该细胞α_(1A)-AR功能的特异性。结果①Hygro-B压力筛选得到58株U2OS-EGFP-NFAT2-α_(1A)-AR细胞,NE 10μmol·L^(-1)孵育后,其中50号细胞核内绿色荧光强度最强,故选定其为稳定共表达α_(1A)-AR和EGFPNFAT2的U2OS-EGFP-NFAT2-α_(1A)-AR细胞。②Western印迹法结果显示,U2OS-EGFP-NFAT2-α_(1A)-AR细胞可明显表达α_(1A)-AR蛋白,而对照细胞U2OS-EGFP-NFAT2中未见α_(1A)-AR蛋白表达。RT-qPCR结果显示,该细胞在传代5~20代内α_(1A)-AR mRNA均稳定表达,其表达水平为对照细胞的500~800倍。③NE或DMED使U2OS-EGFP-NFAT2-α_(1A)-AR细胞中EGFP-NFAT2核转位明显增加,半数有效浓度(EC50)分别为5.94×10^(-7)和6.15×10^(-8) mol·L^(-1)。④与溶剂对照组和萘派地尔组比较,NE组U2OS-EGFP-NFAT2-α_(1A)-AR细胞EGFP-NFAT2核转位明显增强(P<0.01),而萘派地尔+NE组EGFP-NFAT2核转位较NE组明显减弱(P<0.01)。与溶剂对照组和阿替美唑组比较,DMED组EGFP-NFAT2核转位明显增强(P<0.01),阿替美唑+DMED组EGFP-NFAT2核转位与DMED组比较无明显差别,而萘派地尔+DMED组EGFP-NFAT2核转位较DMED组明显减弱(P<0.01)。结论成功构建稳定共表达α_(1A)-AR和EGFP-NFAT2的U2OS-EGFPNFAT2-α_(1A)-AR细胞,可用于靶向α_(1A)-AR化合物筛选和受体分子机制研究。

OBJECTIVE To establish the cells stably co-expressing α_(1A)-adrenergic receptor(α_(1A)-AR)and enhanced green fluorescent protein(EGFP)tagged nuclear factor of activated T cells 2(NFAT2)(EGFP-NFAT2)in U2OS cells.METHODS①The pcDNA3.1-α1-AR-3×FLAG recombinant plasmid was transfected into U2OS-EGFP-NFAT2 cells.The transfected cells were selected by hygromycin B(Hygro-B,200 mg·L^(-1)),and screened by EGFP-NFAT2 nuclear translocation assay after α_(1A)-AR agonist norepinephrine(NE)treatment of 30 min.②The mRNA and protein expression levels of α_(1A)-AR in the selected U2OS-EGFP-NFAT2-α_(1A)-AR cells were examined by real-time quantitative PCR(RT-qPCR)and Western blotting.③U2OS-EGFP-NFAT2-α_(1A)-AR cells were treated with NE(10^(-8)-10^(-5) mol·L^(-1))or dexmedetomidine(DMED,10^(-8.8)-10^(-5) mol·L^(-1)),respectively,for 30 min.EGFP-NFAT2 nuclear translo⁃cation was detected by high throughout screening assay.④The U2OS-EGFP-NFAT2-α_(1A)-AR cells were divided into the solvent control group,α1-AR antagonist naftopidill(1μmol·L^(-1))group,NE(1μmol·L^(-1))group and naftopidill+NE(co-incubation with naftopidill 1μmol·L^(-1) and NE 1μmol·L^(-1))group,α2-AR antagonist atipamezole(ATI,0.1μmol·L^(-1))group,α2-AR agonist DMED(0.1μmol·L^(-1))group,and ATI+DMED(co-incubation with ATI 1μmol·L^(-1) and DMED 0.1μmol·L^(-1))group.The drug incubation time was 30 min.EGFP-NFAT2 nuclear translocation was abserved via a high throughout screening system to validate theα_(1A)-AR function in U2OS-EGFP-NFAT2-α_(1A)-AR cells.RESULTS①There were 58 cell strains expressingα_(1A)-AR in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay.Among these cells,cells No 50 had the highest nuclear translocation function.Theα_(1A)-AR mRNA expression of cells No 50 in 5-20 generations were detected by RT-qPCR and were about 500-800 times that of U2OS-EGFP-NFAT2 cells.②The protein band ofα_(1A)-AR was also detected in cells No 50,but no band ofα_(1A)-AR was detected in U2OS-EGFP-NFAT2 cells by Western blotting.③NE and DMED increased the relative translocation nuclear index in U2OS-EGFP-NFAT2-α_(1A)-AR cells with ED505.94×10^(-7) and 6.15×10^(-8) mol·L^(-1),respectively.④EGFP-NFAT2 nuclear translocation was significant in U2OS-EGFP-NFAT2-α_(1A)-AR cells after NE addition compared with the solvent control or the naftopidill groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the naftopidill+NE group was significantly decreased compared with the NE group(P<0.01).DMED significantly increased the EGFP-NFAT2 nuclear translocation compared with solvent control or the ATI groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the ATI+DMED group was similar to that of the DMED group.The EGFP-NFAT2 nuclear translocation in the naftopidill+DMED group was decreased significantly compared with the DMED group(P<0.01).CONCLUSION U2OS-EGFP-NFAT2-α_(1A)-AR cells stably co-exrepssingα_(1A)-AR and EGFP-NFAT2 are established,which can be used for high throughout screening of biased chemicals and studies on the mechanism ofα_(1A)-AR.