SLC22A12瞬时转染HEK293细胞模型的建立与鉴定
Establishment and identification of SLC22A12 transiently transfected HEK293 cell model

目的:构建SLC22A12瞬时转染HEK293细胞模型并进行鉴定。方法:构建GV142-SLC22A12重组质粒,并优化质粒转染人胚肾细胞HEK293的条件。通过qRT-PCR和Western blot检测SLC-HEK293细胞中SLC22A12 mRNA和hURAT1蛋白的表达情况,借助超高效液相色谱-串联质谱法考察尿酸钠摄取能力。结果:Hind Ⅲ和EcoR Ⅰ双酶切和测序验证显示GV142-SLC22A12质粒构建成功,最佳转染条件为2 μg质粒,试剂(μL):质粒(μg)=3:1。与HEK293细胞相比,SLC-HEK293细胞中SLC22A12 mRNA和hURAT1蛋白表达量上调(P<0.001); 尿酸钠摄取能力更强(P<0.05)。结论:成功建立了SLC22A12瞬时转染HEK293细胞模型。
Aim: To establish and identify a high SLC22A12 expression HEK293 cell line(SLC-HEK293).Methods: Recombinant plasmid GV142-SLC22A12 was constructed and the transfection condition was optimized.The expressions of SLC22A12 mRNA and hURAT1 protein in SLC-HEC293 cell were detected by qRT-PCR and Western blot, and the uptake of sodium urate was investigated by UPLC-MS/MS to confirm the construction of SLC-HEC293.Results: The plasmid of GV142-SLC22A12 was digested by Hind Ⅲ and EcoRⅠ and then was sequenced.The results showed that the GV142-SLC22A12 plasmid was constructed successfully.The optimal transfection conditions was 2 μg plasmid with a 3:1 ratio of agent and plasmid. Compared with HEK293 cell transfected with GV142 plasmid, qRT-PCR and Western blot results showed that the expressions of SLC22A12 mRNA and hURAT1 protein in SLC-HEK293 cells were highly increased(P<0.001), and UPLC-MS/MS results showed that the uptake of sodium urate in SLC-HEK293 was also significantly higher(P<0.05).Conclusion: The transient transfection model of HEK293 cells highly expressing SLC22A12 has been established