CAJ | 학술논문

目的：评价血红素加氧酶-1（HO-1）对 HBV 复制的调控作用。方法采用分子克隆的方法分别构建人 HO-1真核表达载体---pHO 和 HO-1 RNA 干扰质粒---pHi，同 HBV 可复制性克隆 pHBV1．3体外共转染人肝癌细胞系Huh-7，检测 HBV 抗原分泌情况和 HBV 相关 mRNA 含量。利用 HBV 急性感染小鼠模型，腹腔注射钴原卟啉（CoPP）IX和锌原卟啉（ZnPP）IX 分别诱导和抑制 HO-1表达，检测血清 HO-1水平、HBV 效价，免疫组织化学染色观察 HBcAg 在肝细胞内表达情况。分别在细胞水平和动物体内水平，评价 HO-1表达对 HBV 复制的调控作用。结果同空载体共转染细胞相比，pHO 共转染后 HO-1的分泌水平明显上调（犘＜0．01），HBsAg/HBeAg 的分泌受到抑制（均犘＜0．01）；pHi 共转染后 HO-1的分泌水平下降（犘＜0．01），而 HBsAg/HBeAg 分泌增加（犘均＜0．01）；但各组 HBV 相关 RNA 水平无明显差异。CoPPIX 注射后诱导小鼠血清 HO-1高表达（犘＜0．01），ZnPPIX 则有效抑制了 HO-1表达（犘＜0．01）。同对照组相比，CoPPIX 组小鼠血清 HBV DNA 含量降低，肝脏内 HBcAg 阳性染色信号也随之明显减弱（犘均＜0．01）；而 ZnPPIX 组小鼠血清 HBV DNA 含量增加，肝脏内 HBcAg 表达明显增强（均犘＜0．01）。结论上调 HO-1表达可有效抑制 HBV 复制，而下调其表达有利于 HBV 复制，且其可能是在转录后环节上发挥抗 HBV 作用。
목적：평개혈홍소가양매-1（HO-1）대 HBV 복제적조공작용。방법채용분자극륭적방법분별구건인 HO-1진핵표체재체---pHO 화 HO-1 RNA 간우질립---pHi，동 HBV 가복제성극륭 pHBV1．3체외공전염인간암세포계Huh-7，검측 HBV 항원분비정황화 HBV 상관 mRNA 함량。이용 HBV 급성감염소서모형，복강주사고원계람（CoPP）IX화자원계람（ZnPP）IX 분별유도화억제 HO-1표체，검측혈청 HO-1수평、HBV 효개，면역조직화학염색관찰 HBcAg 재간세포내표체정황。분별재세포수평화동물체내수평，평개 HO-1표체대 HBV 복제적조공작용。결과동공재체공전염세포상비，pHO 공전염후 HO-1적분비수평명현상조（마＜0．01），HBsAg/HBeAg 적분비수도억제（균마＜0．01）；pHi 공전염후 HO-1적분비수평하강（마＜0．01），이 HBsAg/HBeAg 분비증가（마균＜0．01）；단각조 HBV 상관 RNA 수평무명현차이。CoPPIX 주사후유도소서혈청 HO-1고표체（마＜0．01），ZnPPIX 칙유효억제료 HO-1표체（마＜0．01）。동대조조상비，CoPPIX 조소서혈청 HBV DNA 함량강저，간장내 HBcAg 양성염색신호야수지명현감약（마균＜0．01）；이 ZnPPIX 조소서혈청 HBV DNA 함량증가，간장내 HBcAg 표체명현증강（균마＜0．01）。결론상조 HO-1표체가유효억제 HBV 복제，이하조기표체유리우 HBV 복제，차기가능시재전록후배절상발휘항 HBV 작용。
Objective To evaluate the regulation of heme oxygenase-1 on HBV replication.Methods Human HO-1 eukaryotic expression vector-pHO and siRNA expression plasmid-pHi were constructed by molecular cloning,respectively. Then they were co-transfected into Huh-7 cell line with HBV replication competent clone-pHBV1 .3 in vitro.Levels of HBsAg and HBeAg in supernatant were detected by ELISA.HBV related mRNA was quantified by RT-PCR.Acute HBV infection mice model were intraperitoneal injected with CoPPIX and ZnPPIX to induce and suppress HO-1 expression in vivo,respectively.Level of HO-1 and expression of HBV from serum,and expression of HBcAg from liver by immunohis-tochemical staining were detected.It was evaluated that the expression of HO-1 regulated HBV replication in vitro and in vivo.Results Compared to control,pHO plasmid co-transfected cells secreted more level of HO-1 (P< 0.01 ),and secretion of HBsAg/HBeAg was inhibited (P<0.01 ).On the contrary,secretion of HO-1 was suppressed significantly (P<0.01)in pHi co-transfected cells,while secretion of HBsAg/HBeAg was increased (P < 0.01 ).No significant difference on HBV related RNA level was observed in different transfected cells.HO-1 expression was induced by CoPPIX injection in mice (P<0.01),while its expression was effectively inhibited by ZnPPIX (P<0.01 ).Compared to control, serum HBV DNA level was lower and positive staining signal of HBcAg in the liver was also decreased obviously in mice treated with CoPPIX (P<0.01 ).Meanwhile,serum HBV DNA and HBcAg expression increased in mice treated with ZnPPIX (P<0.01).Conclusion HBV replication can be effectively suppressed by up-regulating HO-1 expression.On the contrary,HBV replication can be promoted by down-regulating HO-1 expression.The mechanism of HO-1's anti-HBV function probably acts in the post-transcriptional phase in HBV replication life cycle.