基于VEGF/VEGFR2和Dll4/Notch信号通路研究活血化瘀方药对肝癌转移的影响

Blood-activating and Stasis-resolving is of key importance method to treat hepatocellular carcinoma (HCC) , Part of researches consider some blood-activating and stasis-resolving medicinal can stimulate the growth and metastasis of tumors, therefore, promoting blood circulation and removing blood stasis have controversial in treating tumors. Preliminary studies of research group have indicated that the growth and metastasis of tumor was inhibited by the prescription of promoting blood circulation and removing blood stasis, which can keep vascular normalization of tumors, and improve short ‘‘therapeutic window of vascular normalization’’ of angiogenesis inhibitor . Based on the important role of Notch signaling pathways for the growth of blood vessel , we speculate it may work through signal pathway of Notch. So the study proposes the hypothesis that prescription of promoting blood circulation and removing blood stasis would affect metastasis of HCC in prompting tumor vascular normalization by intervening signal pathway of VEGF and Notch. This study adopts implanted hepatoma model of blood stasis, which is intervened by the prescription blood circulation and removing blood stasis , metastasis of tumor are observed following these points, circulating tumor cells are detected by RT-PCR, MVD and MVI are examined in immunohistochemical method, The expression of VEGF, VEGFR2、Notch-4、protein of Dll-4 are detected by using Western blot technology. Based on signal pathway of VERF and Notch, the study will research on effect and mechanism of HCC with blood-activating and stasis-resolving medicine, to provide ideas to solve the controversial problem—use the method of promoting blood circulation and removing blood stasis in treating HCC or not.

活血化瘀法是中医治疗肝癌的重要方法，近年有研究认为某些活血化瘀药能促进肿瘤的生长和转移，活血化瘀法治疗肿瘤存在争议。我们前期研究表明活血化瘀方能促使肝癌血管持续正常化，改善以VEGF为靶点的血管生成抑制剂短暂“血管正常化时间窗”，抑制肿瘤生长和转移。基于Notch信号通路对血管发育的重要作用，我们推测活血化瘀方通过Notch信号通路起作用，因此本研究提出了活血化瘀方通过干预VEGF和Notch信号通路促使肿瘤血管正常化，进而影响肝癌转移的假说。研究通过建立血瘀证移植性肝癌模型，活血化瘀方药进行干预，观察肿瘤转移情况，采用RT-PCR检测循环肿瘤细胞，免疫组化法检测MVD、MVI，Western Blot检测VEGF、VEGFR2、Notch-4、Dll-4蛋白的表达。本研究基于VEGF和Notch信号通路,研究活血化瘀方药对肝癌血管生成的作用和机制，为活血化瘀治疗肝癌的争议提供解决的思路。

活血化瘀法是中医治疗肝癌的重要方法，近年有研究认为某些活血化瘀药能促进肿瘤的生长和转移，活血化瘀法治疗肿瘤存在争议。我们前期研究表明活血化瘀方能促使肝癌血管持续正常化，改善以VEGF为靶点的血管生成抑制剂短暂“血管正常化时间窗”，抑制肿瘤生长和转移。基于Notch信号通路对血管发育的重要作用，我们推测活血化瘀方通过Notch信号通路起作用。研究通过建立血瘀证移植性肝癌模型，活血化瘀方药进行干预。研究发现：（1）瘀血状态下大鼠移植性肝癌生长能力增强，血瘀可能是促进肝癌生长的重要因素。（2）在血瘀状态下，活血化瘀全方及单味药均能降低大鼠移植性肝癌的微血管密度，抑制肿瘤血管生成，促进肿瘤血管正常化。而在非血瘀状态下活血化瘀方能抑制肿瘤微血管的生成，单味药作用不明显，联合应用有协同作用。研究结果表明活血化瘀方能减少肝癌肿瘤微血管的密度，促使肿瘤血管正常化，在血瘀状态下，活血化瘀方以及单味药的作用更显著。（3）在血瘀状态下，活血化瘀方能显著提高大鼠移植性肝癌Notch蛋白的表达水平，降低VEGF、VEGFR2的表达水平，单药三棱、莪术、赤芍单药均能降低VEGFR2蛋白表达水平。而在非血瘀状态下，活血化瘀方及单味药赤芍、三棱对大鼠移植性肝癌血管生成的相关的因子VEGF、VEGFR2、Dll4、Notch蛋白表达无明显作用。结果表明在血瘀状态下，活血化瘀方能抑制VEGF/VEGFR2的表达，减少肿瘤血管的生成，同时提高Notch的表达，促使肿瘤血管正常化，因此，活血化瘀方可能通过激发Notch/Dll4信号通路和抑制VEGF/VEGFR2信号通路发挥抗肿瘤的作用。本研究基于VEGF和Notch信号通路,进一步揭示了活血化瘀方对肝癌血管生成的作用和机制，为活血化瘀治疗肝癌的争议提供解决的思路。