Toll样受体2信号通路在黄曲霉素B1诱导卵圆细胞增殖和恶性转化中的作用及机制研究

A major risk facot for hepatocellular carcinoma(HCC) is aflatoxin ingestion from foods contaminated by the moulds. Worldwide, the incidence of HCC is highest in regions endemic for aflatoxin B1(AFB1). However, the cells arisen of HCC induced by AFB1 are not very well understood. It has been proved that AFB1 exposure produced oval cell proliferation in male Pekin ducklings, rats and woodchucks. Previous works from this laboratory demonstrated that LE/6 cells(the oval cell lines, derived from the adult rats) have the capacity to generate tumors, including HCC, after exposure to the carcinogens. In previos studies, we also found that: (1)AFB1 induced oval cell response in murine models and these oval cells expressed Toll-like receptor 2(TLR2), (2)primary cultured oval cells expressed TLR2, and (3) AFB1 induced the activation of TLR2 signaling pathway. TLR2 activation induces the release of cytokines. There is now a growing body of evidence suggesting that some of these cytokines are involved in development and progression of HCC,and some exert the proliferative effect on oval cells. Based on these previous data, we suggest that TLR2 signaling pathway is involved in proliferation and neoplastic transformation of oval cells induced by AFB1. We would use the murine models developing HCC induced by AFB1 exposure and LE/6 cell lines(oval cell lines) treated with AFB1 in vitro to prove that oval cells give rise to HCC induced by AFB1, study the effects of AFB1 on the biological behaviors of oval cells and explore the roles of TLR2 signaling pathway in proliferation and transformation of oval cells induced by AFB1. Otherwise, we would carry out the studies to determine whether inhibition or silence TLR2 pathway lead to inhibition of adult liver oval cell growth in livers induced by AFB1, and the following HCC development. If these studies accomplished, the data will be useful for us to better understand the oval cell biology and AFB1-induced hepatocarcinogenesis, and help us to develop new potential therapies targeting of TLR2 signaling pathway for HCC.

流行病学资料显示黄曲霉素B1（AFB1）是引起肝细胞癌的主要因素之一。研究提示卵圆细胞可能是肝细胞癌的起源细胞。研究AFB1诱导卵圆细胞增殖和恶性转化的机制，可能有助于阐明肝细胞癌的发生发展及其机制。申请者前期研究发现：AFB1诱导卵圆细胞增殖和恶性转化；AFB1致肝癌小鼠模型中有明显卵圆细胞增殖，增殖卵圆细胞表达Toll样受体2（TLR2）；AFB1激活肝组织中TLR2信号通路。研究已证实TLR2信号通路下游多个因子可以诱导卵圆细胞增殖。基于前期的研究结果，本课题将建立AFB1诱发肝细胞癌的动物模型和作用于肝细胞及卵圆细胞的体外模型，通过体内外实验探讨肝细胞癌的祖细胞起源性，研究AFB1对卵圆细胞增殖分化和恶性转化的影响，探讨TLR2信号通路在此过程中的作用和调控机制。本课题将为肝细胞癌的祖细胞源性提供新的依据，加深肝细胞癌发生及其机制的了解，为肝细胞癌的防治提供新的思路。

肝细胞癌是最常见并且恶性程度最高的肿瘤之一，其细胞起源尚不清楚。研究显示卵圆细胞可能是肝细胞癌的起源细胞，它在致癌物质的作用下可以恶变成肿瘤细胞。黄曲霉素B1是引起肝细胞癌的主要因素之一。研究黄曲霉素B1诱导卵圆细胞增殖和恶性转化的机制，可能有助于阐明肝细胞癌的发生发展及其机制。申请者前期研究发现AFB1诱导小鼠肝内卵圆细胞增殖；应用基因芯片分析发现异常增殖的卵圆细胞内Toll样受体2信号通路有异常表达。本项目在此基础上，进一步研究发现给予黄曲霉素B1后，小鼠肝组织内卵圆细胞明显增殖，肝组织内Toll样受体及其下游因子表达上调；Toll样受体2突变小鼠肝组织内卵圆细胞增殖明显受抑；抑制剂抑制Toll样受体2通路表达，肝组织内卵圆细胞增殖受到明显抑制。在体外模型中，黄曲霉素B1诱导卵圆细胞的增殖，抑制其分化，促进细胞恶变；诱导肝细胞的坏死凋亡。抑制Toll样受体2信号通路，黄曲霉素B1对卵圆细胞的诱导作用明显受到抑制。此外，我们还发现维生素D1通过调控AKT通路抑制黄曲霉素B1诱导卵圆细胞的恶变。本课题为肝细胞癌的祖细胞源性提供新的依据，加深肝细胞癌发生及其机制的了解，为肝细胞癌的防治提供新的思路。