【作者】 徐传瑞；

【导师】 黄士昂；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 研究目的：原癌基因Bmi1在多种肿瘤类型及干细胞中都有表达,然而该基因在肝癌发生以及肝脏干细胞增殖中的作用尚不清楚。本研究试图明确：1.Bmi1在肝癌发生中的作用及其作用机制；2.Bmi1在肝脏干细胞增殖中的作用。研究方法：1.我们使用定量PCR和Western杂交检测了Bmi1肝癌组织中的表达,并使用shRNA在肝癌细胞系中抑制Bmi1的表达,从而观察Bmi1缺失对肝癌细胞系生长的影响。接着我们使用高压注射法转染Bmi1和Ras基因到小鼠中,观察Bmi1是否可以结合其它癌基因诱导肝癌发生。最后,我们结合使用肝癌组织,肝癌细胞系及原代细胞,我们分析了Bmi1是否在肝癌发生中调控Ink4A/Arf途径。2.我们利用化合物DDC在小鼠中诱导卵圆细胞增殖建立小鼠模型,通过比较野生型小鼠和Bmi1敲除小鼠中卵圆细胞增殖的差异来分析Bmi1基因对卵圆细胞增殖的影响。研究结果：1.Bmi1基因在肝细胞肝癌组织及细胞系中都存在过量表达。而当Bmi1的表达被抑制时,细胞生长明显受阻,并且细胞周期不能正常进行。为了明确Bmi1在体内促进肝癌细胞生长的机制,我们稳定表达了Bmi1和／或RasV12,一种Ras的激活形式。我们发现仅有Bmi1或者RasV12不能促进肿瘤在小鼠体内发生。而同时表达Bmi1和Ras V12则可以诱导肿瘤在肝脏中形成。重要的是,我们在体内和体外都没有发现Bmi1抑制Ink4A/Arf的证据。2.Bmi1敲除小鼠中,卵圆细胞,一种可能的肝脏干细胞的增殖存在重要缺陷。在野生型小鼠中,肝脏损失会诱导卵圆细胞大量增殖,而在Bmi1敲除小鼠中,卵圆细胞的增殖数量受到明显抑制。研究结论：1.Bmi1为肝癌肿瘤细胞生长增殖所必需,而且可以结合其它癌基因诱导肿瘤在肝脏中发生,并且其作用机制不依赖于抑制Ink4A/Arf途径；2.Bmi1为卵圆细胞增殖所必需,是介导肝脏干细胞增殖的一个重要基因。更多还原

【Abstract】 Objectives:Bmil is a polycomb family proto-oncogene that has been implicated in multiple tumor types. However its role in HCC development and liver stem cell proliferation has not been well-studied. In this study, we are trying to explore the functions of Bmi1 in hepactic carcinogenesis and hepatic stem cell expansion.Methods:1. We applied qRT-PCR and western blotting to detect the expression of Bmi1 in human HCC tissue. Then we used the shRNA to knock down the expression of Bmi1 in HCC cell lines and to evaluate the impact of loss of Bmi1 on HCC cell growth. To investigate the role of Bmil in promoting liver cancer development in vivo, we stably expressed Bmi1 and/or the activated form of Ras (RasV12) in mouse liver. Mechanism analyis was finished by checking the expression of Ink4A/Arf in human and mice HCC tissues, Bmi1 knock down cell lines and primary mouse hepatocytes.2. To illustrate the role of Bmi1 in maintaining the growth of oval cell, we set up a mouse model. In this mouse model, both Bmi1 WT and KO mice were fed with DDC, a chemical to induce oval cell expansion. By comparing the phenotype of oval cell expansion between Bmi1 WT and Bmi1 KO mice, we can determine the role of Bmi1 in liver stem cell proliferation.Results:1. We found that Bmi1 is over-expressed in human HCC samples as well as HCC cell lines. When Bmi1 expression is knocked down in human HCC cells lines, it significantly inhibits HCC cell proliferation and perturbs cell cycle regulation. In mouse model, while Bmi1 or RasV12 alone is not sufficient to promote liver cancer development, co-expression of Bmi1 plus RasV12 induces HCC formation in mice. Intriguingly, we found no evidence that Bmi1 inhibits Ink4A/Arf expression in both in vitro and in vivo systems of liver tumor development.2. We found that in Bmi1 null mice, the oval cell, a potential hepatic stem cell, showed decreased proliferation capability. In wild type mice, liver injury resulted from DDC treatment is able to induce marked proliferation of oval cells. In Bmil knock-out mice, however, the cell numbers of oval cell induced by liver injury are decreased due to loss of Bmil.Conclusions:1. Bmi1 is required for HCC cell proliferation in vitro and it cooperates with other oncogenes to promote hepatic carcinogenesis in vivo. Bmi1 functions as an oncogene independent of Ink4A/Arf repression in liver cancer development; 2. Bmi1 is required for the proliferation of oval cells and is a potential mediator of oval cell expansion in mouse livers.更多还原