硫氧还蛋白还原系统在肝癌发生发展中的作用：基于黄曲霉素B1诱导肝癌小鼠模型的研究

The cell origin of HCC induced by AFB1 are not very well understood.A major risk facot for hepatocellular carcinoma(HCC) is aflatoxin ingestion from foods contaminated by the moulds. 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. The thioredoxin(Trx) redox system is one of the most important regulators of redox balance and, thus, redox controlled cell functions. Many researches showed that elevated expression of Trx is associated with increased proliferation of tumour cells, inhibition of apoptosis, aggressive tumour growth, and decreased patient survival. Recently, it is showed that Trx system plays an important role in proliferation and differentiation of stem cells. In previous studies, we also found that: (1)AFB1 induced oval cell response in murine models, (2)AFB1 induced the cell death of hepatocytes and had no effect on that of oval cells and (3) the proliferating oval cells expressed high level of Trx. Based on these previous data, we suggest that Trx system is involved in proliferation and differentiation 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 study the effects of AFB1 on the biological behaviors of oval cells and explore the roles of Trx redox systm in proliferation, differentiation and malignant transformation of oval cells induced by AFB1. Otherwise, we would carry out the studies to determine whether inhibiting the expressions of Trx and TrxR 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 Trx system for HCC.

研究提示卵圆细胞可能是肝细胞癌的起源细胞。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。硫氧还蛋白（Trx）还原系统是机体内重要的蛋白还原系统，在肿瘤发生发展和干细胞调控中均起着重要作用。申请者前期研究发现： AFB1诱导卵圆细胞明显增殖，诱导明显的肝细胞凋亡，而对卵圆细胞凋亡无明显影响；增殖的卵圆细胞内有Trx高表达。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，通过体内外实验研究AFB1对卵圆细胞增殖分化和恶性转化的影响，探讨Trx还原系统在此过程中的作用和调控机制。本课题将阐明AFB1诱导卵圆细胞增殖和恶变的部分机制，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。

硫氧还蛋白（Trx）还原系统是机体内重要的蛋白还原系统，在肿瘤发生发展和干细胞调控中均起着重要作用。我们发现黄曲霉毒素B1诱导卵圆细胞ROS产生增加，并激活Trx还原系统，Trx1表达上调，而同时使用ROS的抑制剂NAC处理细胞后，ROS水平及Trx1表达均无明显变化。Trxr1在肝癌组织中的表达水平明显高于癌旁组织，并提示更差的长期生存率。体外实验发现敲减Trxr1可明显抑制肝癌细胞增殖、侵袭和转移，而过表达Trxr1则可促进肝癌细胞增殖、侵袭和转移。体内实验发现抑制Trxr1可明显减弱肝癌细胞的原位成瘤及皮下成瘤能力，同时给予Trxr1抑制剂Auranofin进一步增强此效应。进一步地我们发现Trxr1可激活Akt/mTOR信号通路，从而促使肝癌细胞EMT的发生。另外我们研究Trx1抑制剂PX-12在肝癌细胞中的作用，PX-12可以通过促进凋亡、引起细胞S期阻滞起到抑制肝癌细胞的作用。添加NAC可拮抗PX-12引起的凋亡。PX-12通过引起ROS升高显著增强5-氟尿嘧啶的细胞毒性作用。我们的研究证明了使用硫氧还蛋白系统抑制剂Auranofin或PX-12可在体内抑制肝癌细胞的生长，可能为肝癌的药物治疗提供新的治疗靶点及理论支持。