基于中医伏毒理论的扶正祛毒方对肿瘤干细胞依赖于TF表达及TF/FVIIa信号通路而促进肿瘤转移的干预研究

Cancer stem like cell(CSLCs) is the most important reason of tumors relpase and metastasis,they exist in the body after tumor therapy,and induce the tumors comeback at proper occation,this phenomena is simple to the theory of the "FuDu"of traditional chinese medicine.The theory of the "FuDu" of traditional chinese medicine is to cure the diseas that have not happened, there is little report ahout this theory applying to the prevention and therapy of cancer relapse and metastasis.This reserch based on the TCM "FuDu" theory,and CSLCs high express tissue factor (TF) and TF/FVIIa signal pathway activited, we choose "FuZhengQuDu" formular as the interferon factor; the 4T1 cell line will be the target of this formular in this project.At first we will enrich the CSLCs from 4T1 cell line according to the phenotype of CSCs;secondly ,we will establis servral models which will present the series step of tumor metastasis,;thirdly, we will test the fators which related to the series step of tumor metastasis,such as TGF-β, NKG2D, VEGF, ECM, MMP-9, etc both in vitro and in vivo.In one word,the purpose of this project is to discover the mechanisms of the prevention effect in cancer metastasis by "FuZhengQuDu formular" based on the regulationg effect on CSCs and TF expression as well as the activity of TF/FVIIa signal pathway,then try to provide experimental evidence for the clinic applying "FuZhengQuDu" formular and "FuDu" theroy of TCM.

肿瘤干细胞治疗后长期存活于体内，在适宜时机导致肿瘤复发和转移,这种现象与中医伏毒理论相似。在肿瘤细胞导致肿瘤复发转移的过程中，组织因子(TF)及TF/FVIIa信号通路起着关键的调节作用。中医伏毒理论的根本是治未病，目前基于中医伏毒理论的中医药对肿瘤复发转移的防治研究尚未见报道。本研究基于中医伏毒理论，以肿瘤干细胞高表达TF及活化TF/FVIIa信号通路为研究切入点；以基于伏毒理论的扶正祛毒方为干预措施，以鼠源乳腺癌4T1细胞系为研究对象；分离纯化4T1肿瘤干细胞样细胞（CSLCs）；在体内外建立能反映肿瘤细胞转移系列过程相关的实验模型；采用相关技术进行与肿瘤细胞转移系列过程相关的分子及蛋白的检测。以期从肿瘤干细胞与中医伏毒理论的角度探讨扶正祛毒方对CSLCs的TF表达和TF/FVIIa信号通路的干预作用，为中医药基于伏毒理论对肿瘤干细胞促进肿瘤复发转移的生物学行为的干预调控提供实验依据。

本研究基于乳腺癌干细胞的分离富集纯化，建立了肿瘤细胞自我包被、免疫逃逸、侵袭及促间质生成等实验体系，通过尾静脉接种建立了小鼠血行转移模型。利用RT-PCR、Western Blot、流式细胞仪、高内涵细胞成像、小动物活体成像等技术和方法，观察到乳腺癌干细胞基于高效表达组织因子而活化血小板、逃脱NK细胞免疫监视的作用机制，以及高侵袭、高度促间质生成及促进转移灶形成的生物学行为。同时研究表明，基于“伏毒”理论的扶正祛毒方可能通过减少4T1或乳腺癌干细胞表面的组织因子表达，抑制4Tl或4Tl-CSCs诱导的血小板活化，进一步导致NK细胞杀伤活性增强而达到抑制转移的目的；扶正祛毒方对上皮间质转化的调控作用，可能是通过下调TGF-β/Smad通路以降低间质表型标记蛋白、提高上皮表型标记蛋白来实现的；扶正祛毒方干预血行转移的作用可能与其减少上皮间质转化有关。