CAJ | 학술논문

目的观察高水平的HBV复制对QSG-7701细胞可能产生的致病效应.方法采用磷酸钙沉淀方法转染质粒pUC18-HBV1.2(实验组)和空质粒pUC18(对照组)至QSG-7701细胞,细胞计数观察细胞生长曲线.转染后4 d,采用荧光实时定量PCR方法检测培养上清中HBV DNA水平;免疫荧光细胞化学染色检测细胞内的HBsAg表达;电子显微镜和末端脱氧核苷酸转移酶介导duTP缺口末端标记法(TUNEL)检测细胞凋亡;Oliga信号传导基因芯片检测实验组和对照组的基因差异表达.结果对照组细胞转染6 d后细胞数量增加(8.3±1.2)倍,凋亡细胞极少见.实验组在转染后6 d细胞数量仅增加(1.1±0.2)倍,HBsAg阳性细胞35.4%±6.7%,细胞凋亡占15.2%±4.3%.基因差异表达谱分析显示与细胞生长和凋亡相关的部分基因如CASP3(2.7981)、CASP7(2.2643)、3-Apr(3.5013)、CDC2(0.4380)、MAPK6(0.4447)和MAP3K2(0.2785)等表达水平发生显著改变.结论高水平的HBV复制明显抑制QSG-7701细胞生长,并诱导部分细胞发生凋亡.
목적 관찰고수평적HBV복제대QSG-7701세포가능산생적치병효응.방법 채용린산개침정방법전염질립pUC18-HBV1.2(실험조)화공질립pUC18(대조조)지QSG-7701세포,세포계수관찰세포생장곡선.전염후4 d,채용형광실시정량PCR방법검측배양상청중HBV DNA수평;면역형광세포화학염색검측세포내적HBsAg표체;전자현미경화말단탈양핵감산전이매개도duTP결구말단표기법(TUNEL)검측세포조망;Oliga신호전도기인심편검측실험조화대조조적기인차이표체.결과 대조조세포전염6 d후세포수량증가(8.3±1.2)배,조망세포겁소견.실험조재전염후6 d세포수량부증가(1.1±0.2)배,HBsAg양성세포35.4%±6.7%,세포조망점15.2%±4.3%.기인차이표체보분석현시여세포생장화조망상관적부분기인여CASP3(2.7981)、CASP7(2.2643)、3-Apr(3.5013)、CDC2(0.4380)、MAPK6(0.4447)화MAP3K2(0.2785)등표체수평발생현저개변.결론 고수평적HBV복제명현억제QSG-7701세포생장,병유도부분세포발생조망.
Objective To investigate the effects of high level hepatitis B virus(HBV)replication on the hepatocytes.QSG-7701 cells.Methotis Human hepatocytes of the line QSG-7701 were cultured and transfected with the plasmid pUC18-HBV1.2 or pUCl8 containing 1.2 full length HBV DNA by the standard calcium phosphate precipitation method.Other QSG-7701 cells were transfected with the plasmid pUC18 as controls.Cell growth curves were drawn for 7 days after transfection.Four 4 days after transfeetion,HBV DNA in the culture medium was detected by using fluorescence quantitative real-time PCR.Cell apoptosis was detected by using terminal deoxynucleotidyl transferase.mediated dUTP nick end labeling and electronic microscopy.Differential expressed genes were analyzed by using Oliga signal pathway micro-array.Results The curves of cell growth showed tha the amount of control QSG-7701 cells increased bv(8.3±1.2)times,significantly faster than the pUCl8.HBV1.2 transfected QSG-7701 cells that increased only by(1.1±0.2)times(P＜0.01).Four days after transfection,the HBsAg positive rate of the pUC18-HBV1.2 transfected cells was 35.4%±6.7%.and the apoptotic rate was 15.2%±4.3%.The HBV DNA level in the cuhure supernatant peaked 4 days adder transfection with the maximum value of(5.8±2.6)×106 copies/ml.Genes related to cell growth and apoptosis,such as CASP3(2.7981),CASP7(2.2643),3-Apr(3.5013),CDC2(0.4380),MAPK6(0.4447),and MAP3K2(0.2785),were differentially expressed.Conclusion Higll replicated HBV markedly inhibits the growth of hepatocytes and induces cell apoptosis.