CAJ | 학술논문

目的：探究自噬与乙型肝炎病毒(hepatitis B virus,HBV)复制之间的相互关系。方法利用 HepG2细胞,转染绿色荧光标记的微管相关蛋白轻链3(GFP-LC3),分别在正常和饥饿条件下培养,另共转染 HBV WT 和GFP-LC3,正常条件培养,免疫荧光观察细胞自噬；分别转染1.3 mer HBV 野生型质粒(HBV WT)与 HBV 突变型(HBV X-)质粒,正常和饥饿两种条件下培养,蛋白印迹检测自噬相关蛋白 LC3和乙型肝炎病毒核心蛋白(HBc)的表达；细胞转染 HBV WT 后,分别正常条件培养(对照组),无血清饥饿培养(饥饿组),加入自噬抑制剂3-甲基腺嘌呤(3-MA)培养(3-MA 组),采用酶联免疫吸附测定法(ELISA)检测细胞上清乙型肝炎表面抗原(HBsAg)和乙型肝炎 e 抗原(HBeAg)的含量。结果转染 HBV WT 质粒的细胞内自噬荧光颗粒较对照组明显增加发生自噬的细胞比例分别为13%和2%；饥饿组 HBsAg 含量(1.856±1.415)较对照组(1.531±0.944)升高,HBeAg 含量(0.533±0.391)较对照组(0.334±0.110)升高,差异具有统计学意义(P <0.05)；自噬抑制剂组 HBsAg 含量(1.184±0.759)较饥饿组(1.856±1.415)降低,HBeAg 含量(0.306±0.130)较饥饿组(0.533±0.391)降低(P <0.05)；蛋白印迹显示,转染HBV WT 质粒的细胞中 LC3蛋白表达较 HBV X-组增高,且在饥饿条件下,HBc 蛋白的表达增高。结论乙型肝炎病毒可诱导细胞自噬的发生,且自噬可促进乙型肝炎病毒的复制。
목적：탐구자서여을형간염병독(hepatitis B virus,HBV)복제지간적상호관계。방법이용 HepG2세포,전염록색형광표기적미관상관단백경련3(GFP-LC3),분별재정상화기아조건하배양,령공전염 HBV WT 화GFP-LC3,정상조건배양,면역형광관찰세포자서；분별전염1.3 mer HBV 야생형질립(HBV WT)여 HBV 돌변형(HBV X-)질립,정상화기아량충조건하배양,단백인적검측자서상관단백 LC3화을형간염병독핵심단백(HBc)적표체；세포전염 HBV WT 후,분별정상조건배양(대조조),무혈청기아배양(기아조),가입자서억제제3-갑기선표령(3-MA)배양(3-MA 조),채용매련면역흡부측정법(ELISA)검측세포상청을형간염표면항원(HBsAg)화을형간염 e 항원(HBeAg)적함량。결과전염 HBV WT 질립적세포내자서형광과립교대조조명현증가발생자서적세포비례분별위13%화2%；기아조 HBsAg 함량(1.856±1.415)교대조조(1.531±0.944)승고,HBeAg 함량(0.533±0.391)교대조조(0.334±0.110)승고,차이구유통계학의의(P <0.05)；자서억제제조 HBsAg 함량(1.184±0.759)교기아조(1.856±1.415)강저,HBeAg 함량(0.306±0.130)교기아조(0.533±0.391)강저(P <0.05)；단백인적현시,전염HBV WT 질립적세포중 LC3단백표체교 HBV X-조증고,차재기아조건하,HBc 단백적표체증고。결론을형간염병독가유도세포자서적발생,차자서가촉진을형간염병독적복제。
Objective To investigate the interaction between autophagy and hepatitis B virus replication. Methods HepG2 cells were transfected with GFP-LC3,cultured under normal conditions or starvation.Another co-transfected with wild-type 1.3 mer HBV (HBV WT)and GFP-LC3,normal culture conditions.Autophagy was observed by immunofluorescence.The cells were transfected with HBV WT plasmid and mutant HBV (HBV X-) plasmid,then treated with nutrient starvation.Western blotting was used to analyze the expression of LC3 and HBc. The cells were transfected with HBV WT, cultured with normal conditions (control ),no serum (starvation), autophagy inhibitor (3-MA ), ELISA was used to detect HBsAg and HBeAg of cell supernatants.Results A significant increase of autophagy particles in HBV WT transfected cells (13%)was found compared with that in control group(2%);Titres of HBsAg (1.856±1.41 5)and HBeAg (0.533±0.391)of nutrient starvation group were higher than those of control group (1.531±0.944;0.334±0.1 10)(P <0.05);Titres of HBsAg (1.184±0.759)and HBeAg (0.306±0.130)of 3-MA group were lower than those of starvation group (1.856 ±1.415;0.533 ±0.391)(P <0.05);Western blotting analysis showed that the expression of LC3 in the HBV WT-transfected cells was higher than in mutant group.HBc expression increased in starvation condition.Conclusion Hepatitis B virus can induce cell autophagy.Further more,autophage can enhance HBV DNA replication.