基于Wnt-DKK1信号通路维持肝癌干细胞“干性”探究解毒颗粒预防肝癌术后复发的机制研究

The postoperative recurrence is the main reason for the poor prognosis of liver cancer. Funded by the key projects in the National Science & Technology Pillar Program during the Eleventh Five-year Plan period, our department has performed multi-center randomized clinical trials and confirmed that Jiedu Granule can potently reduce the recurrence rate of liver cancer patients. However, the underlying mechanism has not been well elucidated. . In recent years, enormous results have demonstrated that liver cancer stem cell (LCSC) is the radical reason for the recurrence of liver cancer. We previously performed cellular experiments and confirmed that Jiedu Granule was more efficient in LCSCs than that in non-LCSCs, and it can significantly inhibit the stem-like properties of LCSCs. Moreover, the mRNA gene chip and the relevant experiment results indicated that Jiedu Granule can remarkably inhibited Wnt/β-catenin pathway, and down-regulated the level of DKK1. . It has been determined that Wnt and DKK1 can interact with each other, and then activate the down-streaming β-catenin, which plays an important role in maintaining the stem-like properties of LCSCs. Hence, we hypothesized that the effect of reducing postoperative recurrence rate of liver cancer by Jiedu Granule may be attributed to inhibiting the stem-like properties of LCSCs via Wnt-DKK1 pathway. However, whether the exact relationship between the effect of inhibiting the stem-like properties of LCSCs via Wnt-DKK1 pathway and the effect of reducing postoperative recurrence rate of liver cancer by Jiedu Granule needs more evidence from in vitro and in vivo experiments. Furthermore, the targets and pattern of suppressing Wnt-DKK1 pathway in LCSCs by Jiedu Granule is largely unclear. . Therefore, in this present research, we will utilize a series of new scientific techniques including obtaining LCSCs, in vivo bioluminescence imaging and isolating circulating tumor cells to uncover the mechanism for Jiedu Granule to inhibit the stem-like properties of LCSC by modulating Wnt-DKK1 pathway. Our research will identify the mechanism for the therapeutic effect of liver cancer by Jiedu Granule, providing the theoretical basis for expanding clinical applications of Jiedu Granule in liver cancer treatment.

术后复发是肝癌预后差的主要原因。我科前期在国家“十一五”计划资助下经多中心RCT研究证实：解毒颗粒能降低肝癌术后复发率，但机制不明。肝癌干细胞(LCSC)是肝癌复发的真凶。我们前期细胞学结果显示解毒颗粒对LCSC的敏感性高于非LCSC，并能降低LCSC干性；mRNA芯片及验证结果表明解毒颗粒能抑制Wnt/β-catenin通路、下调DKK1表达。Wnt-DKK1相互影响并激活β-catenin是维持LCSC干性的核心机制。因此，我们提出假说：解毒颗粒抗肝癌术后复发与其调控Wnt-DKK1通路、降低LCSC干性相关。然而，解毒颗粒抑制Wnt-DKK1通路、降低LCSC干性与其抗肝癌术后复发之间的关系尚需要进一步体内外实验确认，且其对Wnt-DKK1通路的作用靶点和机制尚待阐明。本课题拟用LCSC富集、活体成像、CTC分选等技术，揭示解毒颗粒抗肝癌术后复发的机理，为其临床推广应用提供实验依据。

我国是肝癌大国，提高疗效是肝癌研究的“重中之重”。申请者所在单位为国家教育部中西医结合重点学科、国家中医药管理局中医内科（肿瘤）重点学科、上海市中医肝癌专病中心，长期致力于中医药防治肝癌的临床与基础研究。前期在国家科技部“十一五”等科研资助项目下，我们通过多项临床研究证实：本单位创立的中药小复方——解毒颗粒能够有效降低肝癌术后复发率，提高其疗效。然而，解毒颗粒抗肝癌术后复发的机制并不清楚。.最新医学研究认为：干性是肝癌的“核心动力”，Wnt-DKK1相互影响并激活β-catenin是维持LCSC干性的核心机制。我们前期细胞学结果显示解毒颗粒对LCSC的敏感性高于非LCSC，并能降低LCSC干性；mRNA芯片及验证结果表明解毒颗粒能抑制Wnt/β-catenin通路、下调DKK1表达。因此，我们提出假说：解毒颗粒抗肝癌术后复发与其调控Wnt-DKK1通路、降低LCSC干性相关。然而，解毒颗粒抑制Wnt-DKK1通路、降低LCSC干性与其抗肝癌术后复发之间的关系尚需要进一步体内外实验确认，且其对Wnt-DKK1通路的作用靶点和机制尚待阐明。.在本项目的资助下，我们通过培养人肝细胞癌细胞（HCC）、体外富集LCSC以及建立小鼠、裸鼠肝癌模型，证实解毒颗粒能够明显降低肝癌的增殖，降低LCSC干性特性，抑制维持肝癌干性的关键通路——Wnt/β-catenin信号通路，并在细胞总蛋白、mRNA以及细胞上清等多个层面降低DKK1的表达。借助于Wnt/β-catenin信号通路激动剂LiCl以及过表达DKK1的慢病毒载体，我们发现解毒颗粒调控Wnt-DKK1信号通路的主要机制为：解毒颗粒能够直接抑制Wnt/β-catenin经典信号通路，并降低Wnt/β-catenin信号通路的下游蛋白DKK1的表达；解毒颗粒不能直接作用于DKK1，并通过非经典途径抑制Wnt/β-catenin通路。.基于解毒颗粒对维持肝癌干性的关键通路——Wnt/β-catenin通路具有明显的抑制作用，且缺氧微环境是影响肝癌发生发展转归的关键微环境、缺氧微环境能够激活Wnt/β-catenin通路增加肝癌干性，故我们以缺氧诱导的肝癌干性增强为研究方向，进一步发现：解毒颗粒还能在缺氧微环境的条件下通过抑制Wnt/β-catenin信号通路，降低肝癌干性。