基于中医“瘀毒”学说的扶正活血解毒方对肿瘤干细胞依赖于PMNs及异常血液流动状态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 activied polymorphonuclear leukocytes(PMNs) then induced themselves attached to the endothelium cells,transfered to the outside ot the vessels, then achived the metastasis under the abnormal stadus of blood flow. This phenomena is simple to the "YuDu" theory of traditional chinese medicine.There are many clinical evidence indicate that the treatment of "FuZhengHuoXueJieDu" based on the theory of the "YuDu" can prevent the tumor metastasis. But there is little report ahout the mechanism of this prevention effect. This research based on the TCM "YuDu" theory,and CSLCs high express IL-8 and PMNs activited, we choose "FuZhengHuoXueJieDu" 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 establish 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 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 ""FuZhengHuoXueJieDu formular" based on the regulationg effect on CSLCs and IL-8 expression as well as the activity of PMNs,then try to provide experimental evidence for the clinic applying "FuZhengHuoXueJieDu" formular and "YuDu" theroy of TCM.

肿瘤干细胞在机体异常血流状态下，活化中性粒细胞（PMNs），借此与血管内皮细胞粘附并成功地实现血管外渗而导致肿瘤转移,该现象与中医瘀毒学说相似。PMNs及异常血流状态在该过程中起着关键的调节作用。临床上，基于瘀毒学说的中医扶正活血解毒法可以预防肿瘤转移，但目前对其机制及作用靶位研究鲜见报道。本研究基于瘀毒理论，以肿瘤干细胞在异常血流状态下通过IL-8活化PMNs为研究切入点；以扶正活血解毒方为干预措施，以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内皮粘附，血管外渗等转移系列过程相关的实验模型；并采用相关技术进行与肿瘤细胞转移相关的分子及蛋白的检测。以期从肿瘤干细胞与中医瘀毒理论的角度探讨扶正活血解毒方通过对机体血流状态的调节而对CSLCs基于IL-8活化PMNs诱发内皮粘附及外渗的干预作用，为中医药基于瘀毒理论预防肿瘤转移提供实验依据。

本研究基于中医瘀毒理论，以肿瘤干细胞在异常血流状态下通过IL-8活化PMNs诱发内皮粘附及外渗为研究切入点；以扶正活血解毒方为干预措施，以4T1细胞系为研究对象；分离纯化4T1肿瘤干细胞样细胞（CSLCs）；在体内外建立了能反映肿瘤细胞内皮粘附，血管外渗等转移系列过程相关的平行板流动体系、流动迁徙体系、transwell迁移、尾静脉接种肺转移实验模型；研究CSCLs的生物学行为特性；采用相关技术对介导这一过程的关键因子IL-8、ICAM-1、AVB3、整合素β2、t-PA纤溶系统相关分子进行检测。研究结果：1.CSCLs能够分泌更高水平的IL-8，上调粘附分子ICAM-1、AVb3及PMNs表面的整合素β2的表达，具有相较4T1细胞更强的与PMNs聚合、血管内皮细胞粘附及血管外渗的能力。2.扶正活血解毒方能够通过抑制IL-8分泌，降低PMNs膜表面β2整合素表达，抑制4T1细胞、CSCLs与PMNs粘附，抑制血管内皮细胞粘附及血管外渗，从而抑制血行转移。3.扶正活血解毒方能够抑制4T1细胞、CSCLs的迁移，其机制可能与上调肿瘤细胞间粘附分子表达，增强细胞间粘附作用，上调抑癌性分子PAI-1/TFPI表达水平有关。4.在体内实验证实，扶正活血解毒方能够显著抑制小鼠外周血IL-8的水平，进而下调小鼠外周血PMNs整合素β2的表达水平，从而降低肿瘤细胞与PMNs的粘附聚合作用，抑制肿瘤细胞的血行转移。与此同时，扶正祛毒方能够通过上调肿瘤组织细胞表面粘附分子ICAM-1、AVB3的表达水平，增强肿瘤细胞间的粘附作用，稳定瘤体，降低肿瘤的迁移、脱落。5.体内实验动物模型造模过程中，通过提前中药灌喂，激活机体免疫系统的反应，能够获得更好的疗效。本研究从肿瘤干细胞与中医瘀毒理论的角度探讨扶正活血解毒方对CSLCs基于IL-8活化PMNs诱发内皮粘附及外渗促进肿瘤转移的干预作用，从CSLCs与肿瘤细胞招募PMNs，血管内皮粘附，血管外渗以及靶器官种植的角度探讨扶正活血解毒方预防肿瘤复发转移的机制；为中医药特异性靶向调控CSLCs提供理论依据；为扶正活血解毒方预防肿瘤复发转移提供实验依据。