【摘要】 目的:探讨肝细胞内视网膜母细胞瘤(Rb)基因缺陷对转化生长因子-β(TGF-β)诱导的细胞周期静止的影响。方法:分离并培养原代肝细胞,特异性siRNA腺病毒封闭细胞内Rb基因表达后加入TGF-β诱导,然后MTT法检测细胞生长变化,流式细胞仪检测细胞周期变化,并用Western blot和荧光定量逆转录聚合酶反应(FQ-RT-PCR)法检测细胞中pRb、E2F、c-MYC和p16的表达变化。结果:原代培养肝细胞感染Rb-siRNA重组腺病毒后,正常对照肝细胞抑制率高于Rb基因封闭的肝细胞(69.76%,p<0.01),流式细胞检测可见对照组TGF-β诱导后处于G1期的肝细胞数明显增多(99.80%,p<0.05),而在Rb基因封闭组中,TGF-β诱导后各细胞周期分布与未诱导的正常肝细胞基本一致。Western blotting检测可见48h时对照组TGF-β诱导后,E2F和c-MYC蛋白表达减少而p16高表达,Rb基因封闭组TGF-β诱导后c-MYC表达减少而p16表达增高。结论:TGF-β可诱导肝细胞静止于G1期,而Rb基因功能缺陷的肝细胞对TGF-β的诱导敏感性显著降低。更多还原

【Abstract】 Aim:to detect how Rb-deficiency will affect responses to TGF-β induced cell cycle arrest of hepatocyte.Methods:primary hepatocytes were isolated and cultured,and Rb-specific adenovirus siRNA was transformed into cells.Then TGF-β was added daily and cell growth and cell cycles variations.Western blot and FQ-RT-PCR were adopted to detect pRb,E2F,c-MYC and p16 expression changes.Results:Primary hepatocytes were isolated and infected with Rb-specific siRNA adenovirus.In control cells,treatment with TGF-β prevented cells to enter S phase via decreased cMYC activity,activation of P16INK4A activity.In Rb-null hepatocytes,E2F and cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling.Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes.Conclusion:The present results show that otherwise genetically normal hepatocytes with disabled Rb genes respond less well to the antiproliferative effects of TGF-β.更多还原