清胰化积方通过EphrinB1-EphB2轴调控炎性微环境-肿瘤干细胞相互作用逆转胰腺癌耐药机理研究

Our Previous studies found that Qingyihuaji formula(QYHJ) reversed the drug resistance of pancreatic cancer which was more obvious in EphB2 high expression pancreatic cancer cells. QYHJ jointed intervention with Gemcitabine inhibited M2-type polarization of tumor associated macrophages (TAMs) and reduced pancreatic cancer stem cell ratio. The latest research shows that inflammatory microenvironment closely related with pancreatic cancer drug resistance, TAMs are important inflammatory cells, M2 type TAMs induced drug resistance by promoting microvascular abnormalities to generate lowering drug concentration and sensitivity through EphrinB1-EphB2 axle. We put forward our hypothesis according to the above results: QYHJ reverse pancreatic cancer drug resistance by suppressing TAMs M2-type polarization which can promote microvascular normalization to increase drug concentration in pancreatic cancer cells and inhibit cancer stem cells malignant transformation to increased drug sensitivity through EphrinB1-EphB2 axis. This project will establish different EphB2 expression human pancreatic cancer cells, create pancreas orthotopic model established in vivo, build co-culture model of pancreatic cancer cells, endothelial cells and M2-type TAMs in vitro,and reveal the mechanism of reverting drug resistance of QYHJ by explicating the relationship of QYHJ mediated TAMs M2-M1 transformed, microvascular normalization, inhibition of pancreatic cancer stem cells malignant transformation through EphrinB1-EphB2 axis.

前期研究发现清胰化积方具有逆转胰腺癌耐药作用，在EphB2高表达癌细胞中更明显，联合干预抑制肿瘤相关巨噬细胞（Tumor-associated Macrophages，TAMs）M2型极化并降低胰腺癌干细胞比例。最新研究显示炎性微环境与胰腺癌耐药关系密切，TAMs是重要炎性细胞，M2型TAMs通过EphrinB1-EphB2轴促进微血管异常生成降低药物浓度和敏感性诱发耐药。据此提出假说：清胰化积方通过EphrinB1-EphB2轴抑制TAMs M2型极化促进微血管正常增加癌细胞内药物浓度和抑制癌干细胞恶性转化增加药物敏感性发挥作用。项目建立不同EphB2表达人胰腺癌细胞，体内建立原位接种模型，体外建立癌细胞、血管内皮细胞和M2型TAMs共培养模型，通过明确EphrinB1-EphB2轴介导的TAMs M2-M1转化、微血管正常、抑制胰腺癌干细胞转化与逆转耐药关系揭示清胰化积方发挥作用机理。

目的：观察QYHJ方对不同EphB2表达人胰腺癌CFPAC-1裸鼠皮下移植瘤生长抑制作用差异，明确酪氨酸激酶受体EphB2表达与QYHJ方干预是否有效之间的相关性。.方法：QYHJ方抑瘤差异通过建立不同EphB2表达状态人胰腺癌CFPAC-1细胞裸鼠皮下移植瘤模型并以QYHJ方干预四周。采用流式细胞仪分析细胞周期和凋亡变化，进一步分析其抑瘤机理。RT-PCR和Western blot 分别检测相关基因mRNA和蛋白表达变化。.结果：QYHJ方干预四周后，对CFPAC-1、CFPAC-1 Con RNAi细胞抑瘤率分别为31.40%和31.33%，对CFPAC-1 EphB2 RNAi细胞的抑瘤率只有18.36%，QYHJ方对EphB2相对高表达的CFPAC-1细胞表现出更明显的抑制肿瘤生长的作用。细胞周期分析显示QYHJ方干预后，CFPAC-1、CFPAC-1 Con RNi细胞G0/G1期比例分别由67.37%、70.77%上升至76.40%、78.47%，S期细胞由24.70%、22.70%降至17.41%、15.17%，干预组与相应生理盐水组之间G0/G1期差异显著，均有统计学意义（p<0.05，p<0.05），Con RNAi细胞两组S期差异也有统计学意义（p<0.05），而干预前后CFPAC-1 EphB2 RNAi细胞周期并未发生明显变化。干预后CFPAC-1和CFPAC-1 Con RNAi凋亡细胞比例明显增加，分别由干预前的6.21%和4.97%增加至28.03%和20.98%，但统计分析却未显示有统计学意义，干预后CFPAC-1 EphB2 RNAi细胞凋亡并未发生明显变化，凋亡率分别为11.20%和12.53%，提示其对EphB2低表达的细胞凋亡无调控作用。进一步的机理探索发现QYHJ方干预后EphB2 mRNA和蛋白表达并未发生明显变化，但其配体EphrinB1（Eph receptor-interacting B1，EphrinB1）的表达却明显升高，干预后CDK6在CFPAC-1和Con RNAi细胞中明显下调，但在CFPAC-1 EphB2 RNAi细胞并未变化。.结论：QYHJ方对EphB2相对高表达的胰腺癌CFPAC-1细胞抑瘤作用较EphB2低表达的细胞明显，其干预优势部分通过阻滞细胞周期于G0/G1期而抑制细胞增殖。