基于mTOR信号通路探讨健脾解毒方抑制大肠癌血管形成的机制研究

With the living standard raising,the incidence rate of colorectal cancer has presented an ascending tendency in recent years,which is emphasizing the need for improvements in therapeutic options.The 5-year survival rate of surgery,the first-line treatment for colorectal cancer,is just about 50 percent.Traditional Chinese medicine has an important role in the treatment of postoperative and postradiation and after chemotherapy in colorectal cancer. At present, traditional Chinese medicine treatment by Jianpi Jiedu prescription is the main method for colorectal cancer,however,there is a relative lack of accurate mechanism available. Since tumor angiogenesis plays a critical role in the transfer and the occurrence and development of the solid tumors, blocking tumor angiogenesis becomes the main effective means of anti-angiogenic approaches.The abnorm in mTOR signaling pathways, which is a central part of the tumor angiogenesis, is closely related the development of colon cancer. Therefore, mTOR has become the most effective molecular targets for colorectal cancer. To reveal the molecular mechanism of Jianpi Jiedu prescription treatment based on mTOR signaling pathways,we study the influence of Jianpi Jiedu prescription on angiogenesis of colorectal cancer, the effective role composition, function target, the response signal pathway of Jianpi Jiedu prescription by a variety of in vitro and vivo experiments, integrating proteomics, bioinformatics, molecular biology and the cell biology technology,which providing a scientific basis for the standardization of the traditional Chinese medicine.

随着生活水平提高，我国大肠癌发病率逐年上升，因此大肠癌的预治研究具有重要意义。手术仍为大肠癌主要治疗方法，但其根治性手术切除后5年生存率仅为50%左右。中医辨证治疗在大肠癌术后、放化疗后的治疗中有着重要的地位。目前认为健脾解毒法是中医治疗大肠癌的主要方法，但其疗效机制仍需进一步研究。肿瘤血管生成是实体肿瘤发生、发展及转移的关键，阻断血管生成成为治疗恶性肿瘤的主要有效手段之一。mTOR信号通路异常与大肠癌的发生、发展密切相关，是调控肿瘤血管生成的中心环节，已成为治疗大肠癌最有效分子靶点。项目以前期临床及实验研究为基础，以健脾解毒方为实验材料，通过多种体内体外实验，基于mTOR信号通路，集成蛋白组学、生物信息学、分子生物学、细胞生物学等领域的先进技术，研究健脾解毒方对大肠癌血管生成的影响、有效作用成分、作用靶标以及反应信号通路，揭示其分子以及作用机理，为中药的规范化治疗提供科学依据。

健脾解毒法是中医治疗大肠癌的主要方法，但其疗效机制仍需进一步研究。项目基于mTOR信号通路，采用Affymetrix基因表达谱芯片技术，结合基因组学、生物信息学、分子生物学及细胞生物学等领域的先进技术，通过多种体内及体外实验，初步探讨了健脾解毒方对大肠癌血管生成的影响、有效作用成分、作用靶标及反应信号通路。 基于液相色谱、液相色谱-质谱连用的技术检测出健脾解毒方水提取物与冻干粉、含药血清、HCT116细胞、裸鼠肿瘤组织及大鼠肝脏与尿液中多种有效成分及含量。体外及体内研究表明，健脾解毒方能够抑制大肠癌细胞及裸鼠瘤体生长、使细胞周期停滞于G1期、诱导凋亡、抑制侵袭与转移及肿瘤血管形成。健脾解毒方能够下调mTOR mRNA表达，抑制mTOR及其下游信号通路相关蛋白表达、下调与肿瘤血管形成相关蛋白HIF-1α、VEGF及增殖相关的蛋白P53、P21表达。 利用Affymetrix基因表达谱芯片分析健脾解毒方干预HT29细胞后差异表达的基因，采用生物信息学方法，利用IPA软件以mTOR信号通路为中心构建了肿瘤血管形成相关差异表达基因相互作用的反应信号通路图，并用Western印迹验证了关键蛋白质的表达水平。我们的研究结果提示：1、健脾解毒方可能通过mTOR-HIF-1α-VEGF信号通路抑制大肠癌肿瘤血管形成。2、健脾解毒方可能通过mTOR - P53 - P21途径抑制大肠癌生长增殖。本研究为健脾解毒方临床推广应用提供了可靠的实验依据，同时也为中医复方研究提供了较为系统的研究手段与方法，对推进中医药现代化具有较为重要的意义。