CAJ | 학술논문

目的：探讨乙型肝炎病毒（hepatitis B virus, HBV）核心启动子区和前C区突变与肝细胞自噬的关系。方法构建前C区和C启动子区突变的1.3mer HBV质粒G1613A、A 1762T/G1764A、，G1896A；HepG2细胞分别转染GFP-LC3质粒和突变的HBV质粒。免疫荧光染色法检测HBV感染的细胞LC3荧光颗粒表达水平，免疫印迹法分析内源性LC3表达水平，实时定量PCR检测转染后细胞内自噬相关基因Atg5、Atg7和Beclin-1 mRNA的转录水平。结果免疫荧光显示，转染HBV G1613A、A 1762T/G1764A、G1896A质粒的细胞LC3荧光颗粒表达水平分别为（12.5±0.2）%、（11.7±0.2）%、（8.5±0.2）%，低于转染野生型HBV质粒的细胞[（14.5±0.2）%]，差异有统计学意义（P﹤0.05）。实时定量PCR显示，自噬相关基因Atg5 mRNA在转染HBV G1613A、A 1762T/G1764A、G1896A质粒的细胞中的转录水平均明显低于野生型HBV （P﹤0.01），Atg7和Beclin-1在转染G1896A HBV质粒细胞中的基因转录水平低于野生型HBV转染的细胞，且Beclin-1的降低具有统计学意义（P<0.05）。结论 HBV前C区和C启动子区G1613A、A 1762T/G1764A、G1896A突变能够降低HBV感染的肝细胞自噬。
목적：탐토을형간염병독（hepatitis B virus, HBV）핵심계동자구화전C구돌변여간세포자서적관계。방법구건전C구화C계동자구돌변적1.3mer HBV질립G1613A、A 1762T/G1764A、，G1896A；HepG2세포분별전염GFP-LC3질립화돌변적HBV질립。면역형광염색법검측HBV감염적세포LC3형광과립표체수평，면역인적법분석내원성LC3표체수평，실시정량PCR검측전염후세포내자서상관기인Atg5、Atg7화Beclin-1 mRNA적전록수평。결과면역형광현시，전염HBV G1613A、A 1762T/G1764A、G1896A질립적세포LC3형광과립표체수평분별위（12.5±0.2）%、（11.7±0.2）%、（8.5±0.2）%，저우전염야생형HBV질립적세포[（14.5±0.2）%]，차이유통계학의의（P﹤0.05）。실시정량PCR현시，자서상관기인Atg5 mRNA재전염HBV G1613A、A 1762T/G1764A、G1896A질립적세포중적전록수평균명현저우야생형HBV （P﹤0.01），Atg7화Beclin-1재전염G1896A HBV질립세포중적기인전록수평저우야생형HBV전염적세포，차Beclin-1적강저구유통계학의의（P<0.05）。결론 HBV전C구화C계동자구G1613A、A 1762T/G1764A、G1896A돌변능구강저HBV감염적간세포자서。
Objective To investigate the relationship between several common HBV core promoter and pre-core region mutations and hepatocytes autophagy. Methods 1.3mer HBV pre-core and core promoter region mutated vector G1613A, A1762T/G1764A, G1896A were constructed. HepG2 cells were co-transfected with GFP-LC3 plasmid and mutated HBV vector, then LC3 punctas in HBV infected cells were counted by Immunofluorescence. Endogenous LC3 protein were detected by Western Blotting. mRNA levels of autophagy related gene Atg5, Atg7 and Beclin-1 were tested by realtime-PCR. Results Compared with wild type HBV vector [(14.5 ±0.2)%], LC3 punctas in mutated HBV G1613A,A 1762T/G1764A,G1896A vector decreased to (12.5 ±0.2)%, (11.7 ±0.2)%, (8.5 ±0.2)%respectively (P all<0.05). The transcription of autophagy related gene Atg5 in mutated HBV G1613A,A 1762T/G1764A, G1896A vector was less than wild type HBV (P<0.01). Atg7 and Beclin-1 in mutated HBV G1896A vector were less than wild type HBV. Conclusion Pre-core and core promoter mutation (G1613A,A 1762T/G1764A,G1896A) of HBV could decrease autophagy of HBV infected hepatocytes.