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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 Tollip on the secretion of HBsAg and HBeAg in human hepatocellular carcinoma cell line HepG2215 and to find out a new target for the treatment of hepatitis B virus. Methods The recombinant plasmid pCMV-HA-Tollip containing HA tag was identified by double digestion with EcoRⅠand KpnⅠ, then transfected into HepG2215 cells by LipofectamineTM2000. Four transfection groups were established: Lipofectamine transfection control group (15μl (3μg plasmid, 3μg pCMV-HA-Tollip, 15μl liposome), and the recombinant plasmids were transfected into control group (6μg empty plasmid, 15μl liposome), empty plasmid recombinant plasmid co-transfection group After transfection for 48 h, the cells were harvested and the expression of AKT, p-AKT, NF-κB and HA tag protein were detected by Western blot. The supernatants were collected and the ELISA Method to detect the level of HBsAg and HBeAg. Results The recombinant plasmid pCMV-HA-Tollip was identified by double enzyme digestion. The expression of HA tagged protein was observed in both empty vector plasmid transfected group and recombinant plasmid transfected group, but no HA tag protein was detected in the transfected group and empty plasmid transfected control group. Compared with empty plasmid transfected control group Compared with the control group, the expressions of NF-κB and p-AKT protein were significantly decreased (P <0.01), while the expression of AKT protein did not change significantly (P> 0.05); empty The contents of HBsAg and HBeAg in the supernatant of plasmid transfected group and recombinant plasmid transfected group were significantly lower than those of empty plasmid transfected control group (P <0.01), and the inhibitory rates of HBsAg were 24% and 41% The inhibition rates on HBeAg were 13% and 31%, respectively. Conclusion The recombinant plasmid pCMV-HA-Tollip can effectively inhibit the secretion of HBsAg and HBeAg in HepG2215 cells. The mechanism may be related to the inhibition of AKT phosphorylation and down-regulation of NF-κB expression.