【摘要】 目的观察地塞米松处理人肝癌细胞HepG₂对细胞内源性NTCP mRNA的表达影响,以及刺激后该体系在抗HBV药物筛选中的应用。方法采用不同浓度地塞米松刺激HepG₂细胞,处理后不同时间提取HepG₂细胞中总RNA,实时荧光定量PCR检测NTCP mRNA表达量,确定刺激NTCP mRNA表达最佳浓度及时间。采用最佳浓度和时间刺激HepG₂细胞,洗去培养基中残留地塞米松,实时荧光定量PCR检测NTCP mRNA表达变化。用HepG₂.2.15细胞培养上清浓缩HBV感染地塞米松刺激后的HepG₂细胞,ELISA检测细胞培养上清中HBs Ag。最后,采用阳性药物验证该体系的有效性、可行性。结果 0.5μm地塞米松刺激HepG₂细胞24h时NTCP mRNA表达量最高。撤去地塞米松后96h内,HepG₂细胞中内源性NTCP mRNA的表达有一定程度的下降并维持在一定水平。HBV感染地塞米松刺激后的HepG₂细胞后,在培养上清中能够检测到乙肝表面抗原。在该体系中,阿德福韦酯抗HBV活性的EC50为0.26μm,环孢菌素A的EC₅₀为0.19μm。结论地塞米松能够上调HepG₂细胞内源性NTCP mRNA的表达,能够更有效地介导HBV感染,且可以应用于抗HBV药物筛选。更多还原

【Abstract】 Objective Aim to observe the effects of dexamethasone on the expression of endogenous NTCP mRNA in HepG₂ and whether it could use for screening anti-HBV compounds. Methods The total RNA was extracted from HepG₂ cells which were stimulated by different concentrations of dexamethasone for different time. The expression of NTCP mRNA was detected by q PCR. Under the optimized condition,q PCR detected the expression after washing out dexamethasone. Dexamethasone stimulated HepG₂ was infected by HBV which enriched from supernatant of HepG₂. 2. 15,and then detected HBs Ag by ELISA. As a standard,Adefovir Dipivoxil and Ciclosporin A verified the feasibility of this system. Results Stimulated by 0. 5μM dexamethasone for 24 h,the expression level of NTCP mRNA in HepG₂ reached the peak and kept at a high level after washing out dexamethasone within 96 h. The HBs Ag has been detected in HBV infected the dexamethasone stimulated HepG₂. In this system,the EC₅₀ s of Adefovir Dipivoxil and Cyclosporin A are 0. 26 M and0. 19μM,respectively. Conclusion The endogenous NTCP in HepG₂ could be up-regulated by dexamethasone. This system could be infected by HBV. It could use as a system for screening anti-HBV compounds.更多还原