中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
8期
614-618
,共5页
麦丽%杨林%邝建玉%朱建芸%康艳红%张富程%谢奇峰%高志良
麥麗%楊林%鄺建玉%硃建蕓%康豔紅%張富程%謝奇峰%高誌良
맥려%양림%광건옥%주건예%강염홍%장부정%사기봉%고지량
细胞周期%基因,p16%甲基化%乙型肝炎病毒X蛋白
細胞週期%基因,p16%甲基化%乙型肝炎病毒X蛋白
세포주기%기인,p16%갑기화%을형간염병독X단백
Cell cycle%Genes,p16%Methylation%Hepatitis B virus X protein
目的 探讨乙型肝炎病毒X蛋白(HBx)对肝癌细胞生长的影响及其机制.方法 以HepG2细胞和稳定表达GFP/HBx融合蛋白的HepG2/GFP-HBx为实验细胞,四甲基偶氮唑盐(MTT)法检测细胞吸光度值A490,分析细胞生长增殖；流式细胞术检测细胞周期；甲基化PCR检测细胞p16INK4A基因的甲基化；Western blot检测p16蛋白表达水平. 结果 72 h时HepG2/GFP-HBx细胞组的A490为3.225±0.038,分别与HepG2和HepG2-GFP细胞组的2.012±0.022、2.038±0.029比较,增殖能力明显增强,t值分别为46.86和42.51,P值均＜0.001,差异均有统计学意义.流式细胞术检测示HepG2/GFP-HBx细胞组的G0/G1期细胞占16.45％±0.45％,明显低于HepG2和HepG2/GFP细胞组的44.81％±1.36％、42.76％±1.58％,t值分别为-34.22和-28.88,P值均＜ 0.001,差异均有统计学意义.而5-氮-2 '-脱氧胞苷(5-Aza-CdR)处理的HepG2/GFP-HBx细胞G0/G1期细胞比例为33.25％±0.79％,较未处理HepG2/GFP-HBx细胞组的16.45％±0.45％显著增加,两组比较,t=31.85,P＜0.001,差异有统计学意义.甲基化PCR检测显示HepG2/GFP-HBx细胞组发生了p16INK4A基因甲基化,而HepG2和HepG2-GFP细胞组及5-Aza-CdR处理的HepG2/GFP-HBx细胞组未检测到p16INK4A基因启动子甲基化.Western blot检测示HepG2/GFP-HBx细胞组p16蛋白水平低于HepG2和HepG2/GFP细胞组,而5-Aza-CdR处理HepG2/GFP-HBx细胞后,p16蛋白水平高于未处理的HepG2/GFP-HBx细胞. 结论 HBx蛋白可通过缩短HepG2细胞生长的G0/G1期而加快细胞周期进程,从而促进肝癌细胞生长增殖,其机制可能与HBx蛋白诱导p16INK4A基因启动子甲基化及抑制p16蛋白表达有关.
目的 探討乙型肝炎病毒X蛋白(HBx)對肝癌細胞生長的影響及其機製.方法 以HepG2細胞和穩定錶達GFP/HBx融閤蛋白的HepG2/GFP-HBx為實驗細胞,四甲基偶氮唑鹽(MTT)法檢測細胞吸光度值A490,分析細胞生長增殖；流式細胞術檢測細胞週期；甲基化PCR檢測細胞p16INK4A基因的甲基化；Western blot檢測p16蛋白錶達水平. 結果 72 h時HepG2/GFP-HBx細胞組的A490為3.225±0.038,分彆與HepG2和HepG2-GFP細胞組的2.012±0.022、2.038±0.029比較,增殖能力明顯增彊,t值分彆為46.86和42.51,P值均＜0.001,差異均有統計學意義.流式細胞術檢測示HepG2/GFP-HBx細胞組的G0/G1期細胞佔16.45％±0.45％,明顯低于HepG2和HepG2/GFP細胞組的44.81％±1.36％、42.76％±1.58％,t值分彆為-34.22和-28.88,P值均＜ 0.001,差異均有統計學意義.而5-氮-2 '-脫氧胞苷(5-Aza-CdR)處理的HepG2/GFP-HBx細胞G0/G1期細胞比例為33.25％±0.79％,較未處理HepG2/GFP-HBx細胞組的16.45％±0.45％顯著增加,兩組比較,t=31.85,P＜0.001,差異有統計學意義.甲基化PCR檢測顯示HepG2/GFP-HBx細胞組髮生瞭p16INK4A基因甲基化,而HepG2和HepG2-GFP細胞組及5-Aza-CdR處理的HepG2/GFP-HBx細胞組未檢測到p16INK4A基因啟動子甲基化.Western blot檢測示HepG2/GFP-HBx細胞組p16蛋白水平低于HepG2和HepG2/GFP細胞組,而5-Aza-CdR處理HepG2/GFP-HBx細胞後,p16蛋白水平高于未處理的HepG2/GFP-HBx細胞. 結論 HBx蛋白可通過縮短HepG2細胞生長的G0/G1期而加快細胞週期進程,從而促進肝癌細胞生長增殖,其機製可能與HBx蛋白誘導p16INK4A基因啟動子甲基化及抑製p16蛋白錶達有關.
목적 탐토을형간염병독X단백(HBx)대간암세포생장적영향급기궤제.방법 이HepG2세포화은정표체GFP/HBx융합단백적HepG2/GFP-HBx위실험세포,사갑기우담서염(MTT)법검측세포흡광도치A490,분석세포생장증식；류식세포술검측세포주기；갑기화PCR검측세포p16INK4A기인적갑기화；Western blot검측p16단백표체수평. 결과 72 h시HepG2/GFP-HBx세포조적A490위3.225±0.038,분별여HepG2화HepG2-GFP세포조적2.012±0.022、2.038±0.029비교,증식능력명현증강,t치분별위46.86화42.51,P치균＜0.001,차이균유통계학의의.류식세포술검측시HepG2/GFP-HBx세포조적G0/G1기세포점16.45％±0.45％,명현저우HepG2화HepG2/GFP세포조적44.81％±1.36％、42.76％±1.58％,t치분별위-34.22화-28.88,P치균＜ 0.001,차이균유통계학의의.이5-담-2 '-탈양포감(5-Aza-CdR)처리적HepG2/GFP-HBx세포G0/G1기세포비례위33.25％±0.79％,교미처리HepG2/GFP-HBx세포조적16.45％±0.45％현저증가,량조비교,t=31.85,P＜0.001,차이유통계학의의.갑기화PCR검측현시HepG2/GFP-HBx세포조발생료p16INK4A기인갑기화,이HepG2화HepG2-GFP세포조급5-Aza-CdR처리적HepG2/GFP-HBx세포조미검측도p16INK4A기인계동자갑기화.Western blot검측시HepG2/GFP-HBx세포조p16단백수평저우HepG2화HepG2/GFP세포조,이5-Aza-CdR처리HepG2/GFP-HBx세포후,p16단백수평고우미처리적HepG2/GFP-HBx세포. 결론 HBx단백가통과축단HepG2세포생장적G0/G1기이가쾌세포주기진정,종이촉진간암세포생장증식,기궤제가능여HBx단백유도p16INK4A기인계동자갑기화급억제p16단백표체유관.
Objective To investigate the effects and related mechanisms of hepatitis B virus X (HBx)protein on cell cycle and growth in hepatocellular carcinoma.Methods A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment,and HepG2 parental and HepG2/GFP cells was used as the controls.Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of ceils with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L).Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.Results The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225 ± 0.038 A490) than either control (HepG2:2.012 ± 0.022 A490,t =-46.86,P ＜ 0.001; HepG2/GFP:2.038 ± 0.029 A490,t =42.51,P ＜ 0.001).The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45％ ± 0.45％) than either control (HepG2:44.81％ ± 1.36％,t =-34.202,P ＜ 0.001; HepG2/GFP:42.76％ ± 1.58％,t =-28.88,P ＜ 0.001).However,5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25％ ± 0.79％,t =31.85,P ＜ 0.001).The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells,and became demithylation after treatment with 5-Aza-CdR.However,no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells.The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs.HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.Conclusion HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase,and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p 16 protein expression.
