HDAC3缺失抑制小鼠肝再生并引起肝细胞凋亡

Ablation of histone deacetylase 3 in hepatocytes inhibits liver regeneration and induces apoptosis in mice

目的:检测组蛋白去乙酰化酶3(histone deacetylases 3,HDAC3)的缺失对部分肝切除(partial hepatectomy,PH)后肝细胞的增殖、细胞周期和凋亡的影响.方法:通过HDAC3^(flox/flox)小鼠和他莫昔芬(tamoxifen,TAM)诱导的ALB-CRE^(ERT)工具鼠杂交,获得TAM诱导肝脏中HDAC3敲除小鼠(ALB-CRE^(ERT)HDAC3^(flox/flox)mice).Western blot和组织学分析分别检测TAM诱导的ALB-CRE^(ERT)HDAC3^(flox/flox)小鼠肝脏中HDAC3的敲除效率以及组织学改变.通过70%PH构建肝再生模型,并在相应时间点取材.溴脱氧尿苷(5-Bromo-2-deoxyuridine,BrdU)掺入实验和Ki67免疫组织化学染色评估肝细胞增殖情况.Western blot检测细胞周期蛋白(cyclins)和细胞周期蛋白依赖性激酶(cyclin-dependent-kinases,CDKs)的表达水平.原位缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL)和凋亡标志物Cleaved Caspase3蛋白表达水平反映肝细胞的凋亡.结果:TAM诱导的ALB-CRE^(ERT)HDAC3^(flox/flox)小鼠(HDAC3~△小鼠)肝脏中HDAC3表达水平显著降低,但其肝脏结构和代谢并不发生明显改变.BrdU和Ki67免疫组织化学染色结果显示70%PH后36h和48 h肝细胞增殖率明显降低Western blot显示HDAC3~△小鼠cyclin B和cyclin D表达水平显著降低而CDK2和CDK4的表达水平无显著差异,组织学及Western blot均提示肝再生期间肝细胞凋亡增多.结论:HDAC3在小鼠肝再生中起至关重要的作用,HDAC3的缺失抑制肝再生并引起肝细胞凋亡.

AIM：To investigate the role of histone deacetylase 3（HDAC3） in liver regeneration followed by partial hepatectomy（PH）.METHODS：HDAC3 floxed mice were crossed with a tamoxifen-inducible ALBCRE^（ERT） allele to generate inducible tamoxifendependent liver-specific HDAC3 knockout mice（ALB-CRE^（ERT）HDAC3^（flox/flox） mice）.The efficiency of HDAC3 recombination and histological changes after tamoxifen treatment in adult ALB-CRE^（ERT）HDAC3^（flox/flox） mice were determined.Liver regeneration was induced by a 70%PH and mice were sacrificed at various time points after surgery.5-Bromo-2-deoxyuridine（BrdU） incorporation and Ki67 immunohistochemistry were performed to observe the mitotic progression.The expression of mitotic markers cyclins and cyclin-dependent-kinases（CDKs） was detected by Western blot.Apoptosis of hepatocytes was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling（TUNEL） assay and immunoblotting of cleaved Caspase3.RESULTS：HDAC3 in tamoxifen-treated ALB-CRE^（ERT）HDAC3^（flox/flox） mice（HDAC3~△mice） was efficiently deleted.HDAC3~△ mice did not show any apparent changes in liver histology or metabolism compared to control mice without TAM injeciton.The restoration of remnant liver mass was significantly reduced in HDAC3~△ mice.Dramatic lower hepatocyte mitotic figures in HDAC3△mice determined were observed at 36-48 h after PH as indicated by BrdU and Ki67 immunohistochemistry staining.Western blot analysis confirmed that deletion of HDAC3 resulted in poor expression of cyclin B and cyclin D,whereas CDK2 and CDK4 were kept similar both in control and HDAC3~△ mice at36 h and 48 h after PH.In addition,noticeably increased apoptotic hepatocytes were found in HDAC3~△ livers.CONCLUSION：HDAC3 plays crucial roles in the regulation of liver regeneration,and loss of HDAC3 inhibits liver regeneration and induces apoptosis.