摘要
目的探讨Tollip转染人肝癌细胞系HepG2215细胞对HBsAg和HBeAg分泌的影响及其机制,为乙型肝炎的治疗寻找新的靶点。方法含HA标签的重组表达质粒pCMV-HA-Tollip经EcoRⅠ和KpnⅠ双酶切鉴定后,采用脂质体LipofectamineTM2000转染HepG2215细胞,共设4个转染组:脂质体转染对照组(15μl脂质体)、空质粒转染对照组(6μg空质粒、15μl脂质体)、空质粒重组质粒共转染组(3μg空质粒、3μg pCMV-HA-Tollip、15μl脂质体)及重组质粒转染组(6μgpCMV-HA-Tollip、15μl脂质体),转染48 h后,收集细胞,经Western blot检测AKT、p-AKT、NF-κB以及HA标签蛋白的表达;收集上清,ELISA法检测HBsAg和HBeAg的水平。结果重组质粒pCMV-HA-Tollip经双酶切鉴定构建正确。空质粒重组质粒共转染组和重组质粒转染组均有HA标签蛋白的表达,而脂质体转染对照组和空质粒转染对照组无HA标签蛋白表达;与空质粒转染对照组比较,空质粒重组质粒共转染组和重组质粒转染组中NF-κB和p-AKT蛋白的表达明显降低(P<0.01),而AKT蛋白的表达无明显变化(P>0.05);空质粒重组质粒共转染组和重组质粒转染组细胞上清中HBsAg和HBeAg的含量明显低于空质粒转染对照组(P<0.01),对HBsAg的抑制率分别为24%和41%,对HBeAg的抑制率分别为13%和31%。结论 pCMV-HA-Tollip重组质粒在HepG2215细胞中能有效抑制HBsAg和HBeAg的分泌,其机制可能与抑制AKT的磷酸化和下调NF-κB的表达有关。
Objective To investigate the effect of transfection of human liver cancer HepG2215 cells with Tollip on secretions of HBsAg and HBeAg as well as the relevant mechanism,and to search a novel target for therapy of hepatitis B.Methods Recombinant plasmid pCMV-HA-Tollip with a HA tag was digested with EcoRⅠ and KpnⅠ,and transfected to HepG2215 cells in mediation of LipofectamineTM 2000.HepG2215 cells were divided into liposome control,empty plasmid control,empty plasmid ＋ pCMV-HA-Tollip and pCMV-HA-Tollip groups,and transfected with 15 μl LipofectamineTM2000,6 μg empty plasmid ＋ 15 μl LipofectamineTM 2000,3 μg empty plasmid ＋ 3 μg pCMV-HA-Tollip ＋ 15 μg LipofectamineTM 2000 and 6 μg pCMV-HA-Tollip ＋ 15 μl LipofectamineTM 2000,respectively.Forty-eight hours after transfection,the cells were collected and determined for expressions of AKT,p-AKT,NF-κB and HA tag by Western blot,while the supernatant was collected and determined for HBsAg and HBeAg levels by ELISA.Results Restriction analysis proved that recombinant plasmid pCMV-HA-Tollip was constructed correctly.HA tag protein was expressed in empty plasmid ＋ pCMV-HA-Tollip and pCMV-HA-Tollip groups,while was unexpressed in liposome control and empty plasmid control groups.Compared with those in empty plasmid control group,the expression levels of NF-κB and p-AKT in empty plasmid ＋ pCMV-HA-Tollip and pCMV-HA-Tollip group decreased significantly（P 0.01）,while that of AKT showed no significant change（P 0.05）.The HBsAg and HBeAg levels in empty plasmid ＋ pCMV-HA-Tollip and pCMV-HA-Tollip groups were significantly lower than those in empty plasmid control group（P 0.01）.The inhibiting rates to HBsAg in empty plasmid ＋ pCMV-HA-Tollip and pCMV-HA-Tollip groups were 24% and 41%,while those to HBeAg were 13% and 31%,respectively.Conclusion Recombinant plasmid pCMV-HA-Tollip inhibited the secretions of HBsAg and HBeAg in HepG2215 cells effectively by a mechanism which might be associated with inhibiting the phosphorylation of AKT and down-regulating the expression of NF-κB.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第12期1587-1590,共4页
Chinese Journal of Biologicals
基金
重庆市自然科学基金(CSTC 2011lljjA10020
CSTC 2009BB5064)
重庆医科大学基础医学院青年基金(0800280018)
