Objective : To investigate the role of wild type Notch signaling in human hepaocarcinogenesis. Methods: The correlations and biological effects of Notch1 and Ras were analyzed by introducing recombinant vectors NICD-pEGFP-C1 and/or H-Ras-pEGFP-C1 to human non-tumor hepatic cell line L02 cells. The viability of L02 cells transfected with recombinant vector was performed by MTT and the proliferation of L02 cells was tested using clone assay. The transformation of L02 cells was evaluated in vivo. Results: MTT assay show that forced overexpression of Notch1-ICD and H-Ras promoted proliferation and growth of unmanipulated L02 cells( P ＜ 0. 01 ).Cells treated with DAPT showed a significant reduction in cell viability compared with controls( P ＜0. 01 ). Constitutively active Notch1 alone failed to do so by transform immortalized L02 cells in vivo, it synergized with the Ras pathway to promote hepatic cells transformation( P ＜ 0. 01 ). Conclusion : We show that Ras- Notch signaling cooperation drives hepatic cells transformation and stimulates proliferation. Dysregulated Notch signaling cooperation with the Ras in transformation and proliferation offers combined inhibition of the two pathways as an attractive avenue for therapeutic intervention for this advanced disease.%目的:研究失调的Notch 信号与癌基因ras共同作用对正常肝细胞转化和增殖的影响.方法:构建Notch1、Ras的重组载体NICD-pEGFP-C1、H-Ras-pEGFP-C1,利用重组载体共转染正常肝细胞系L02,用MTT法及克隆形成实验检测细胞增殖能力,行体内致瘤实验了解细胞转化状况.结果:MTT检测结果显示,外源性Notch1-ICD能促进L02细胞的增殖,与癌基因ras共转染能显著增加L02细胞的增殖;γ-分泌酶抑制剂DAPT处理共转染的L02细胞,结果显示,随DAPT浓度增加细胞增殖活性呈依赖性降低;克隆形成实验显示,共转染外源性的Notch1胞内结构域NICD和Ras能促进L02细胞克隆形成;体内致瘤实验显示,Ras与Notch1-ICD共同作用能促进L02细胞转化.结论:失调的Notch 信号与癌基因ras共同作用促使正常肝细胞转化和增殖,从而导致肝细胞恶性转变,同时抑制Notch 信号、Ras两条信号转导通路可能是肝癌治疗的新策略.
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