姜黄汤抑制ERK1/2信号通路影响大肠癌干细胞干性和增殖、侵袭、转移的机制研究

We have found that ERK1/2 signal pathway was active in colorectal cancer stem cells(CR-CSCs) and CD133 positive colorectal cancer tissues. Based on the results, firstly we will Inhibit ERK1/2 signal pathway to find the effect of this pathway on stemness, tumor growth, invasion and metastasis, and to analyze the proteins expression related to this pathway. Secondly we will observe the role of traditional chinese medicine--curcuma decoction in mataining stemness and malignant characteristics of CR-CSCs. Then we will establish an subcutaneous xenotransplanted tumor model and liver metastatic model of human colorectal carcinoma in nude mice in order to observe the effect of curcuma decoction and ERK1/2 inhibitor in vivo, meanwhile, detect the expression of the stem markers and the key factors associated with proliferation and apoptosis in the xenograft and liver metastatic cancer tissues by immunohistochemistry techniques and plot survival curves. Finally, disclose the effect of ERK1/2 signal pathway in inhibiting CR-CSCs stemness, tumor growth, invasion and metastasis and the interventional effect of curcuma decoction on this pathway, elucidate the molecular mechanism of curcuma decoction in CR-CSCs. We aim to provide a basis for future molecular biology experiments to find effective target of interfering CR-CSCs with traditional Chinese medicine, as well as new lines of thinking and methods in the treatment theory of managing colorectal cancer.

基于前期研究结果，ERK1/2信号通路在大肠癌干细胞及CD133高表达的大肠癌组织中异常活化。我们拟首先观察特异性抑制ERK1/2信号通路对大肠癌干细胞干性及增殖、侵袭、转移等生物学行为的影响，同时分析下游相关因子的差异表达。引入中医经验方姜黄汤，观察其对大肠癌干细胞恶性生物学行为的影响及对ERK1/2信号通路的干预作用。构建裸鼠肠癌皮下移植瘤及肝转移瘤模型，观察姜黄汤及ERK1/2抑制剂对模型鼠体内肿瘤的抑制作用，免疫组化检测皮下移植瘤及肝转移瘤组织中p-ERK1/2、大肠癌干细胞干性标志及与肿瘤增殖、凋亡相关蛋白表达情况，绘制裸鼠生存曲线。分析总结ERK1/2信号通路在大肠癌干细胞干性维持、增殖、侵袭、转移过程中的作用及姜黄汤对该通路的干预作用，揭示中药姜黄汤作用于大肠癌干细胞的分子机制，为寻找姜黄汤干预大肠癌干细胞的有效靶点提供实验依据，同时也为丰富大肠癌治法理论提供新的思路与方法。

本研究的目标为深入分析和探讨中药经验方姜黄汤影响CR-CRCs恶性生物学行为的可能作用机制，将为姜黄汤治疗大肠癌的临床应用奠定更为可靠的研究基础和理论支持。经过三年的资助研究，我们建立了更成熟的CR-CRCs培养体系，拥有了稳定传代培养的CR-CRCs细胞株。以此为基础完成了系列研究工作。利用基因组学研究分析CR-CRCs中差异表达的基因及相关信号通路并进行生物学验证。证实ERK1/2信号通路与CR-CRCs恶性生物学行为的相关性。体外实验观察姜黄汤对CR-CRCs生物学行为的影响，发现姜黄汤可抑制显著抑制CR-CRCs增殖并促进其凋亡。最后通过裸鼠移植瘤体内实验，进一步证实姜黄汤及其有效成分姜黄素可抑制移植瘤的生长。经药物干预后小鼠移植瘤组织的中干性标志物及增殖相关蛋白表达下降，凋亡相关蛋白表达明显升高。同时检测结果显示小鼠移植瘤组织中p-ERK1/2表达上调，揭示姜黄汤可能通过激活ERK1/2信号通路而发挥其抗肿瘤作用。令人兴奋的是在基础实验过程中我们发现抑癌基因STK11在CR-CRCs中表达下调，而经姜黄素干预后的CR-CRCs中STK11表达上调，因此提出实验假设姜黄汤通过STK11这个关键基因激活ERK1/2信号通路发挥其抑瘤作用，尚需后续实验证实。本研究内容为姜黄汤的临床应用提供了一定的立论依据。更重要的是在此研究基础上仍可进行后续研究，有望深层次探究中医药抑制恶性肿瘤的具体机制。