CAJ | 학술논문

目的：建立大鼠嘌呤能受体 P2 X4（rP2 X4）稳定表达细胞系，并对细胞系功能进行验证。方法构建绿色荧光蛋白rP2X4受体（pEGFP-N1-rP2X4）重组质粒，采用脂质体转染法将pEGFP-N1-rP2X4转染于人胚肾（HEK293）细胞，采用抗生素G418（1 g·L-1）压力筛选，qRT-PCR和Western蛋白质印迹法验证rP2 X4受体的表达量。全细胞膜片钳技术检测稳定表达的 rP2 X4受体Ca2＋电流。结果测序结果表明， pEGFP-N1-rP2X4重组质粒序列完全正确；采用脂质体法稳定转染HEK293细胞后，qRT-PCR和Western蛋白质印迹法结果表明，HEK293-pEGFP-N1-rP2X4稳定转染细胞系在连续传代25代后（1，3，5，10，15，20和25代）rP2X4受体均保持稳定表达；全细胞电流记录实验表明，嘌呤受体激动剂ATP 3．0μmol·L-1能激活HEK293-pEGFP-N1-rP2X4细胞上表达的 rP2X4受体并产生特异性激活电流，且该电流能被非选择性P2X4受体拮抗剂三硝基苯酚（TNP）-ATP（30．0μmol·L-1）阻断。结论 HEK293-pEGFP-N1-rP2X4稳定转染细胞系上rP2 X4受体表达稳定，激活后能产生特异性激活电流。该细胞系可用于rP2 X4受体的药物筛选及其机制研究。
목적：건립대서표령능수체 P2 X4（rP2 X4）은정표체세포계，병대세포계공능진행험증。방법구건록색형광단백rP2X4수체（pEGFP-N1-rP2X4）중조질립，채용지질체전염법장pEGFP-N1-rP2X4전염우인배신（HEK293）세포，채용항생소G418（1 g·L-1）압력사선，qRT-PCR화Western단백질인적법험증rP2 X4수체적표체량。전세포막편겸기술검측은정표체적 rP2 X4수체Ca2＋전류。결과측서결과표명， pEGFP-N1-rP2X4중조질립서렬완전정학；채용지질체법은정전염HEK293세포후，qRT-PCR화Western단백질인적법결과표명，HEK293-pEGFP-N1-rP2X4은정전염세포계재련속전대25대후（1，3，5，10，15，20화25대）rP2X4수체균보지은정표체；전세포전류기록실험표명，표령수체격동제ATP 3．0μmol·L-1능격활HEK293-pEGFP-N1-rP2X4세포상표체적 rP2X4수체병산생특이성격활전류，차해전류능피비선택성P2X4수체길항제삼초기분분（TNP）-ATP（30．0μmol·L-1）조단。결론 HEK293-pEGFP-N1-rP2X4은정전염세포계상rP2 X4수체표체은정，격활후능산생특이성격활전류。해세포계가용우rP2 X4수체적약물사선급기궤제연구。
OBJECTIVE Toestablisharecombinantcelllinethatcanstablyexpressratpurinergic P2receptorP2X4(rP2X4R).METHODS ToconstructgreenfluorescentproteinandrP2X4recombinant plasmid (pEGFP-N1-rP2X4),lipofectamine was used to transfect pEGFP-N1-rP2X4 into human embry-onic kidney (HEK293)cells that were screened with G41 8 (1 g·L-1 ).The quantitative expression of rP2X4 receptor was verified by qRT-PCR and Western blotting analysis.Whole-cell patch clamp record-ing was used to investigate the function of the stably expressed rP2X4 receptor. RESULTS The sequence of plasmid pEGFP-N1-rP2X4 was verified by PubMed Blastn comparison.qRT-PCR and Western blotting analysis demonstrated that the expression of rP2X4 receptor in HEK293-pEGFP-N1-rP2X4 cell lines remained stable after 25 generations (P1 ,P3,P5,P10,P15,P20 and P25).Whole-cell patch clamp recording experiments showed that the rP2X4 receptor agonist,purine-5′-triphosphate (ATP,3.0 μmol·L-1 ),could activate rP2X4 receptors in HEK293-pEGFP-N1-rP2X4 cell lines.Specific activating current could be blocked by non-selective rP2X4 receptor antagonist TNP-ATP (30.0μmol·L-1).CONCLUSION rP2X4receptorisstablyexpressedinHEK293-pEGFP-N1-rP2X4cell line and maintains stable expression and function within 25 continuous generations.The establish ment of HEK293-pEGFP-N1-rP2X4 cell line can contribute to further investigations of the roles of rP2X4 receptors in neuropathic pain.