基于Notch信号通路调控肠癌干细胞样特性探讨消痰散结方抗肠癌侵袭转移的作用机制

Colon cancer stem cells have the features of highly tumorigenic, multi-differentiation, self-renewal and unlimited proliferation. These features play important role in the invasion and metastasis of colorectal cancer. Abnormal activation of the Notch signaling pathway is the critical pathway to effect the biological behavior of colon cancer stem cells. The project team put forward that "phlegm" is the essence of invasion and metastasis in colorectal cancer when we combined with long-term clinical practice. The treatment of “sputum theory of traditional Chinese medicine” is one of the effective method to control gastrointestinal tumor. Xiaotan Sanjie Recipe is the representative recipe of “sputum theory of TCM”. Previous studies showed that Xiaotan Sanjie Recipe had clearly effect in colon cancer, prevent the proliferation of colon cancer stem cells in experimental studies and regulated the expression of Oct4、Nanog. We verified that Notch signaling pathways was high expression in specimens of colorectal liver metastases in clinical. Xiaotan Sanjie Recipe could reduce the expression of Notch1 in specimens of colorectal liver metastases. According to this, we propose that Xiaotan Sanjie Recipe can possibly resist the biological characteristics of cancer stem cells through the regulation of Notch signaling pathway which can achieve the purpose of resistance to invasion and metastasis of colorectal cancer. We will enrich spheroid cells through culturing colorectal cancer cells in Stem Cell Media. We will observe the influence of the biological characteristics of cancer stem cells and the expression of the proteins in Notch signaling pathway through the treatment of Xiaotan Sanjie Recipe in vitro by blocking and activation Notch signaling pathway. Meanwhile, we will prepare tumor-bearing mouse model of colorectal liver metastases by using spheres cells to observe the influence of colon cancer stem cells in the sample characteristics and the proteins in Notch signaling pathway using Xiaotan Sanjie Recipe in vivo. The project is going to state the part of mechanism that Xiaotan Sanjie Recipe can resist to invasion and metastasis of colorectal cancer at the levels of molecule, cells and animals. At the same time, we will clarify the mechanism in the treatment of “sputum theory of traditional Chinese medicine” through the project.

肠癌干细胞样特性与肠癌侵袭转移关系密切，异常激活的Notch信号通路是肠癌干细胞发挥其干细胞样特性的关键途径。项目组结合长期临床实践，提出“痰”为肠癌侵袭转移之本质，“从痰论治”肠癌为有效治法之一。代表方消痰散结方用于肠癌临床疗效明确，前期基础研究发现，其可抑制肠癌干细胞增殖，下调干性标志物Oct4、Nanog表达；临床已验证肠癌肝转移标本中Notch信号通路高表达，该方可下调Notch1表达。我们提出消痰散结方可能通过调节Notch通路及其下游基因表达，抑制肠癌干细胞样特性抗肠癌侵袭转移的假说。项目拟采用SFM富集肠癌干细胞，阻断及激活Notch通路，体外观察该通路与肠癌干细胞样特性的关系及消痰散结方的干预作用；借助肠癌干细胞建立肠癌肝转移荷瘤鼠模型，体内观察该方对肠癌干细胞样特性及Notch通路的影响，从分子、细胞、整体水平上阐明中药“从痰论治”抗肠癌侵袭转移的理论依据及部分作用机制。

结直肠癌是常见的消化系统恶性肿瘤，在全世界范围内其发病率和死亡率居首位，侵袭转移是晚期结直肠癌患者预后差和死亡的主要原因。因此研究结肠癌侵袭转移的发生机制、探索其有效的治疗方法具有重要的临床意义。肠癌干细胞样特性与肠癌侵袭转移关系密切，异常激活的Notch信号通路是肠癌干细胞发挥其干细胞样特性的关键途径。项目组结合长期临床实践，提出“痰”为肠癌侵袭转移之本质，“从痰论治”肠癌为临床有效治法之一。其代表方消痰散结方用于肠癌临床疗效明确，本课题运用无血清悬浮培养系统SFM培养亲代HCT116细胞，富集成球，结果发现其体外增殖能力和体内致瘤能力均强于亲代细胞，球体细胞中部分结肠癌干细胞标志物、细胞表面特异性抗原表达上调，证实球体细胞具有肠癌干细胞样特性。同时观察发现球体细胞中Notch通路关键蛋白Notch1及下游靶基因Hes1蛋白表达均显著上调，经通路抑制剂DAPT及激动剂FC干预，进行阻断或异常激活Notch通路时，HCT116球体细胞增殖、侵袭、迁移等恶性行为均呈现不同程度的下调或增强，证实Notch信号通路的异常激活是HCT116肠癌干细胞发挥其干细胞样特性的关键途径之一。进一步体外实验发现消痰散结方可抑制结肠癌干细胞增殖、侵袭、迁移，阻滞细胞周期、诱导细胞凋亡、下调肠癌干细胞标志物Oct4、Nanog，下调该通路特征性的受体蛋白Notch3、配体蛋白Jagged1、下游基因Hes1及EMT标志物Snail表达。体内实验经裸鼠皮下注射球体细胞造模，消痰散结方可抑制结肠癌干细胞移植瘤的生长，并能在体内下调部分结肠癌干细胞标志物，下调Nocth通路受体蛋白Nocth2、配体蛋白Jagged1、Jagged2、下游基因Hes1及EMT标志物Twist1的表达。本项目可基本证实消痰散结方通过抑制肠癌干细胞样特性实现抗结肠癌侵袭转移的作用，其机制可能与调控Notch信号通路，下调该通路特征性的受体配体蛋白分子、下游基因表达Hes1及EMT标志物相关。本项目深化了消痰散结方在临床对结肠癌侵袭转移的分子机制认识，为阐释中医“从痰论治”肠癌的科学原理提供数据支持。