α2B-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共表达稳转细胞株的构建

Establishment of stably-transfected cell line with α2B-adrenergic receptor and enhanced green fluorescent protein labeled nucleus factor of activated T cells 2

目的建立能够稳定表达α2B-肾上腺素能受体(α2B-AR)和增强型绿色荧光蛋白(EGFP)标记的活化T细胞核因子2(NFAT2)(EGFP-NFAT2)的稳转细胞株。方法构建具有潮霉素(Hydro)抗性的pcDNA3.1α2B-AR重组质粒(pcDNA3.1-Hygro-α2B-AR),转染至表达EGFP-NFAT2的U2OS细胞(U2OS-EGFP-NFAT2细胞),使用Hygro 200 mg·L-1压力筛选10 d后,进行NFAT2核转位实验筛选细胞株并用Z’因子法评价细胞模型的可靠性,采用实时定量PCR和Western蛋白印迹法,检测α2B-AR的mRNA和蛋白表达水平,选择α2-AR的激动剂盐酸右美托咪啶(DMED)和拮抗剂盐酸阿替美唑(ATI)通过NFAT2核转位实验评价α2B-AR的功能活性。结果成功构建重组质粒pc DNA3.1-Hygro-α2B-AR后,稳定转染至U2OS-EGFP-NFAT2细胞,采用NFAT2核转位实验筛选发现,编号12的细胞株相对核转位指数为0.445,活性最高;3次独立实验中,Z’因子分别为0.664,0.533和0.634。实时定量PCR结果显示,编号12的细胞株α2B-AR mRNA表达水平是U2OS-EGFP-NFAT2细胞株的140倍,表达显著增加(P<0.01),且在20代次内表达稳定。Western蛋白印迹结果显示,编号12的细胞株出现明显的α2B-AR蛋白条带,而U2OS-EGFP-NFAT2细胞中未检测到α2B-AR蛋白表达。DMED可以浓度依赖性地提高该细胞株的核转位水平[EC50=(2.616±0.121)nmol·L-1],ATI可以浓度依赖性地对抗DMED的作用[IC50=(89.05±0.22)nmol·L-1]。结论成功构建了共表达α2B-AR与EGFP-NFAT2的稳转细胞株,可用于靶向α2B-AR化合物的筛选和受体分子机制的研究。

OBJECTIVE To establish a stably-transfected cel line with α2B-adrenergic receptor(α2B-AR)and enhanced green fluorescent protein(EGFP) labeled nucleus factor of activated T cells 2(NFAT2)(EGFP-NFAT2) in U2 OS. METHODS U2 OS cells that stably expressed EGFP-NFAT2(U2 OS-EGFPNFAT2 cells) were transfected with pcDNA3.1 expressing hygromycin B(Hygro) resistance gene andα2B-AR gene(pcDNA3.1-Hygro-α2B-AR) recombinant plasmid. The transfected cells were selected by Hygro(200 mg · L-1) for 10 d before being screened on the nucleus translocation assay of EGFPNFAT2. Then, the selected cells were evaluated by Z′ factor for functional stability and α2B-AR expression in the selected cells was analyzed by quantitative real-time PCR(qRT-PCR) and Western blotting.Finally, the activity of α2B-AR on NFAT2 nucleus translocation was evaluated by α2-AR agonist dexmedetomidine hydrochloride(DMED) or antagonist atipamezole hydrochloride(ATI) treatment. RESULTS Recombinant plasmid pcDNA3.1-Hygro-α2B-AR was established and transfected to U2 OS cells stably expressing EGFP-NFAT2. In the stably-transfected No.12 cell strain, the relative nuclear translocation index(0.445) was the highest among the selected cell lines, and Z′ factors on three independent experiments were 0.664, 0.533 and 0.634, respectively. The mRNA expression of α2B-AR of No.12 cell strain was detected by qRT-PCR and was about 140 times that of U2 OS-EGFP-NFAT2 cells(P<0.01)in 20 generations. The protein band of α2B-AR was also detected in No.12 cell strain whereas no band of α2B-AR was detected in U2 OS-EGFP-NFAT2 cell by Western blotting. DMED concentration-dependently increased the relative translocation nuclear index in U2 OS-EGFP-NFAT2-α2B-AR cell 〔EC50=(2.616±0.121) nmol·L-1〕. ATI concentration-dependently decreased the relative nuclear translocation index in U2 OS-EGFP-NFAT2-α2B-AR cell〔IC50=(89.05±0.22) nmol·L-1〕. CONCLUSION The stablytransfected U2 OS-EGFP-NFAT2-α2B-AR cell line is established and can be used for high throughout screening of biased chemicals and the study on the mechanism of α2B-AR.