黄芩苷调节上皮间质转化抑制结肠癌干细胞增殖、侵袭的作用与机制研究

The cancer stem cells (CSCs) are the underlying cause of colon cancer to chemotherapy-resistant, metastasis and relapse. Solid tumors stem cells gain self-renewal, aggressive traits and chemoresistance by epithelial to mesenchymal transition (EMT) after TGF-β signaling pathway activation. The inhibition of invasion and metastasis through inhibition of the EMT of CSCs received extensive attention in recent years. The experiments performed by our group have confirmed that Baicalin not only has the ability to induce colon cancer cells apoptosis, but also has a significant inhibitory effect on the growth and metastasis of nude mouse orthotopic xenografts model of colorectal cancer. The mechanism of these actions is related to proteins that down-regulate the expression of TGF-β、PCNA、P53 genes. In this study, flow cytometry, real-time quantitative RT-PCR, Western bloting and other techniques will be used to further observe the effect of Baicalin on the CSCs proliferation, differentiation and invasion ability in vivo and in vitro. The aim of this study is to explore the effect of Baicalin on the TGF-β/Smads pathway and TGF-β-induced EMT as possible molecular mechanisms that mediate self-renewal and differentiation of CSCs. This research will be not only an endeavor to look for new target and drug for colon cancer therapy, but also a new exploration on the effective component of traditional Chinese medicine.

结肠癌肿瘤干细胞是导致结肠癌对放化疗耐药和/或转移复发的根本原因。TGF-β信号通路的激活促使实体瘤干细胞上皮间质转化后获得了增殖、侵袭能力，增加了对化疗药物的耐药性。近年来，阻断肿瘤干细胞上皮间质转化，抑制其增殖、分化和侵袭引起广泛关注。本课题组前期研究证实了黄芩苷能够诱导结肠癌肿瘤细胞凋亡，具有明显抑制结肠癌裸鼠原位移植瘤生长和转移的作用，其机制与下调TGF-β、PCNA、P53等基因表达有关。本研究拟进一步从体外观察黄芩苷干预对结肠癌肿瘤干细胞增殖、分化和侵袭能力的影响，并通过裸鼠原位移植瘤体内实验，使用流式细胞术、荧光定量、免疫印迹等方法，探讨黄芩苷通过TGF-β/Smads信号通路调控上皮间质转化抑制结肠癌干细胞增殖、分化和转移潜能的分子机制，寻找结直肠癌特异性治疗靶点和药物，同时也是对中药有效成分作用机制的新探索。

黄芩苷是一种来自中药黄芩（Scutellaria baicalensis Georgi）的黄酮类化合物，虽然据报道在各种癌症中具有抗肿瘤活性，但其分子机制仍然知之甚少。本研究旨在探讨黄芩苷抑制结直肠癌细胞生长和细胞迁移/侵袭，诱导细胞凋亡的分子机制。.体外培养结肠直肠癌细胞系，包括RKO，HCT116和SW480用于研究黄芩苷对结直肠癌抑制作用。应用蛋白质印迹，流式细胞仪检测，集落形成和CSC球体形成等方法检测细胞凋亡和细胞周期/生长抑制，EMT（上皮细胞向间充质细胞转变）和细胞干性；Transwell实验用于确定黄芩苷对癌细胞迁移和侵袭的影响。在体内，建立绿色荧光转染的HCT116 肿瘤细胞的裸鼠结肠原位移植肿瘤模型，通过黄芩苷干预，应用IHC和蛋白质印迹实验来探讨黄芩苷诱导的结肠直肠癌细胞生长抑制和分子机制。.黄芩苷不仅可抑制结直肠癌细胞的内源性EMT，还可通过抑制TGFβ/ Smad通路逆转外源性TGFβ1诱导的细胞EMT。黄芩苷治疗结直肠癌细胞诱导G1期细胞周期停滞并引发细胞凋亡。此外，黄芩苷阻断了肿瘤干细胞（CSC）特性的获得，并抑制了结直肠癌细胞中CSC样细胞球的形成。在体内，黄芩苷通过下调EMT，细胞周期和CSC标志物的蛋白质水平，在结直肠癌细胞的原位移植肿瘤模型中进行抗肿瘤作用。 .与SW480细胞（错配修复基因正常）相比，黄芩苷具有特异性抑制RKO细胞（错配修复基因hMLH1甲基化）和HCT116细胞（错配修复基因hMLH1缺失）的作用，提示黄芩苷可能是错配修复基因功能缺陷的结直肠癌的有效治疗药物。