基于血管拟态及PI3K通路探讨肝癌索拉非尼耐药机制及益气化瘀解毒方干预作用研究

Resistance of sorafenib which targeted on Liver cancer is the problem to be solved in the treatment of liver cancer. Sorafenib can block the liver cancer angiogenesis, which cause local hypoxia, and liver cancer cells in hypoxia environment can get blood supply by vascular mimicry, this has close relationship with the resistance. Research shows that the activation of PI3K pathway mediates the liver cancer cells of sorafenib drug resistance, and key factors of vascular mimicry formation:Bcl-2、MMP-2、MMP-9、OPN involved. So, It is expected to obtain a new breakthrough to study on the mechanism of Sorafeni resistance in liver cancer based on vascular mimicry and PI3K pathway.Previous studies confirmed that YiQiHuaYuJieDu decoction can inhibit the growth of nude mouse's transplantation tumor, and has a regulatory role of VM and key factor on the above. Based on this basis, we design experiment in vitro and in vivoto,to further explore molecular regulation mechanism of the Sorafenib resistance in liver based on vascular mimicry and PI3K signaling pathways. to clarify effect of YiQiHuaYuJieDu decoction on liver cancer resistance and key targets,to provide new ideas and scientific basis for developing more new drugs of Chinese medicine for Chinese medicine reverse Sorafenib resistance.

肝癌分子靶向药物索拉非尼耐药是目前肝癌治疗中亟待解决的问题。索拉非尼能够阻断肝癌血管新生，造成局部缺氧，而肝癌细胞在缺氧环境下可通过血管拟态获取血液供应，血管拟态可能为肝癌索拉非尼耐药的关键原因。研究表明，PI3K通路的激活介导了肝癌细胞对索拉非尼的继发性耐药，且与血管拟态形成关键因子HIF1a、Bcl-2、MMP-2、MMP-9、OPN有密切关系。因此围绕血管拟态及PI3K通路研究肝癌索拉非尼耐药有望获得新的突破。我们前期研究证实益气化瘀解毒方能抑制人肝癌细胞和裸鼠移植瘤的生长，并对血管拟态及上述关键因子具有调控作用。本项目拟在此基础上，设计体外、体内实验，深入探讨血管拟态及PI3K信号通路在肝癌索拉非尼耐药的分子调控机制，阐明益气化瘀解毒方对肝癌索拉非尼耐药的作用及关键靶点，为中医药逆转Sorafenib耐药和开发抗肝癌多靶点的中药新药提供新的思路和科学依据。

当今，深入探讨肝癌索拉非尼耐药对于改善肝癌治疗预后具有重要意义，其机制可能与肝癌血管拟态和PI3K /Akt /mTOR通路有关。本研究拟通过体内外实验，明确益气化瘀解毒方对人肝癌Sorafenib耐药细胞和裸鼠移植瘤的作用，阐明肝癌血管拟态及PI3K /Akt /mTOR通路在肝癌索拉非尼耐药的机制。. 本研究发现，人肝癌索拉非尼耐药细胞QGY7702/Sora与非耐药细胞MRP、GST- 和Topo Ⅱ三基因表达水平并无显著异。益气化瘀解毒方可显著降低GST-的表达；益气化瘀解毒方联合Sorafenib可提高细胞的死亡率，可促进TopoⅡ表达。耐药细胞与不耐药细胞相比，E-Cad低表达，VEGF、HIF1a、Bcl-2高表达，益气化瘀解毒方联合Sorafenib能促进E-Cad表达，抑制VEGF、HIF1a、Bcl-2的表达。细胞实验证实了益气化瘀解毒方对耐药细胞的抑制作用，并且证实其对血管拟态相关因子E-Cad、VEGF、HIF1a、Bcl-2表达的影响。实验建立了耐药细胞株的裸鼠移植瘤模型，索拉非尼对移植瘤增长无抑制作用，益气化瘀解毒方联合索拉非尼对移植瘤具有抑制作用，说明耐药移植瘤模型是成功的。免疫组化检测显示耐药细胞移植瘤HIF1a、VM表达高于非耐药细胞移植瘤组织，说明耐药与血管拟态有关，益气化瘀解毒方联合Sorafenib能降低其表达。Real-time PCR检测显示耐药细胞和非耐药细胞瘤组织AKT、OPN、Bcl-2、VEGF、CXCL12、CXCR4、STAT3基因的表达存在差异，中西药联合干预可显著降低CXCR4、JAK基因的表达，其作用机制可能与此有关。