基于Wnt/β-catenin和CXCR4探讨细辛醚调控结肠癌干细胞干性维持及转移潜能的机制研究

The cancer stem cells(CSCs) are reported in the colon cancer by more and more literatures, which may be the cause of chemotherapy resistance,metastasis and/or relapse.The anti-CSCs research is drawing scientists' attention. Acorus calamu is a triditional Chinese medicine and the anti-cancer research about its essential oil, asarone was limited. Our group have performed the experiments that showed the asarone has the ability to induce the clone cancer LoVo cells apoptosis through the mitochondrial pathway. We further confirmed that asarone can inhibit the colons and sphere formation. We plan to sort the colon CSCs in vitro and observe the effect of asaron on the cells proliferation, differentiation and invasion ability. We also want to use the nude mice to establish a xenograft model and a liver metastasis of colon cancer model. The real-time PCR, western blotting and immunohistochemistry technologies were explored to check the key molecular changes in the Wnt/β-catenin and CXCL12/CXCR4 pathways. This project was aimed to examine the anti-cancer effect of asarone in the CSCs aspect and to explore the molecular mechanism of asarone in inhibiting self-renewal and inducing differentiation. This research will be not only a useful exploration on the effective conponent of triditional Chinese medicine, but also a endeavor to look for new drugs for the colon cancer therapy.

近年来，文献报道结肠癌中存在肿瘤干细胞，其可能是导致肿瘤的耐药和/或转移复发的根本原因。针对肿瘤干细胞的药物研究正引起科学家们的广泛关注。石菖蒲是传统中药，而其挥发油成分中的主要物质细辛醚的抗肿瘤研究鲜有报道。本课题组前期实验证实了其单体α-细辛醚能通过线粒体途径体外诱导结肠癌LoVo细胞的凋亡，进一步发现其能抑制细胞克隆形成和肿瘤干样细胞悬浮球的形成。本研究拟从体外分选结肠癌干细胞，观察细辛醚干预对其增殖、分化、侵袭能力的影响，并通过裸鼠移植瘤和结肠癌肝转移瘤的体内实验，使用实时荧光定量、免疫印迹、免疫组化等实验，检测Wnt/β-catenin和CXCL12/CXCR4通路中关键分子的改变。本项目从肿瘤干细胞角度探讨细辛醚的抗肿瘤作用，并从抑制自我更新及诱导分化的角度研究该化合物可能的分子机理，既是对传统中药有效成分作用机制的一项新的探索，同时也努力为结肠癌治疗寻求新的药物研究的方向。

本研究项目首先通过体内体外实验确定了细辛醚诱导结肠癌细胞凋亡的机制之一是通过线粒体途径；接着课题组花费了较大的精力来确定结肠癌干细胞的表面标志为CD133highCD44high，证明了此群细胞是结肠癌细胞的干细胞样细胞，其多处于细胞周期的静止期G0/G1，高表达干性相关基因，低表达细胞分化相关基因，具有更强的增殖潜能，并能在体内成瘤实验中显示出更强的成瘤能力。之后按计划进行了动物的体内实验，使用两种不同的造模方法，皮下移植瘤和结肠癌细胞经脾肝转移模型，观测在体实验中细辛醚的作用，发现细辛醚对结肠癌皮下移植瘤有抑制作用，同时有减少结肠癌细胞经脾肝转移的趋势。但在体外干细胞实验过程中，细辛醚对于CD133highCD44high细胞增殖的干预作用有限，并且其对于Wnt/β-catenin和CXCL12/CXCR4信号通路的影响效果与预期有差距，提示细辛醚的抑瘤作用还有其他的信号通路。