Cyr61调控肿瘤相关巨噬细胞-胰腺癌细胞交互对话促进胰腺癌肝转移作用机制及中药干预研究

Pancreatic cancer with liver metastases patients have poor prognosis and lack of effective treatment so far, to find new therapeutic target will have important clinical value. Our previous project found that QYHJ inhibit liver metastases of pancreatic cancer by down-regulating Cyr61 and regulating EMT, follow-up study also found QYHJ can inhibit tumor microenvironment macrophages (TAM) infiltration and IL-6 secretion. Therefore, we propose the hypothesis: QYHJ down-regulated the expression of Cyr61 regulates EMT and inhibited pancreatic cancer with liver metastasis may rely on the tumor micro-environment TAM participation, Cyr61 may be regulation feedback loop of tumor associated macrophages and pancreatic cancer cells to promote the growth and metastasis of pancreatic cancer cells. To verify this hypothesis, we intend to use liposome transfection and co-culture system of pancreatic cancer cells and macrophages, to make clear mechanism of Cyr61 regulating crosstalk of tumor associated macrophages and pancreatic cancer cells, to promote pancreatic cancer cells growth and metastasis; through QYHJ intervention in vivo and in vitro studies, to clarify the anti-metastatic molecular mechanism. From the new point of view of Cyr61regulating crosstalk of tumor associated macrophages and pancreatic cancer cell, this study will provide new ideas and new therapeutic targets of pancreatic cancer with liver metastasis treated by traditional Chinese medicine.

胰腺癌肝转移患者预后差，迄今尚缺乏有效治疗手段，寻找新的治疗靶点将具有重要临床价值。课题组前期面上项目证实清胰化积方通过下调Cyr61调节EMT抑制胰腺癌肝转移，后续研究中我们又发现清胰化积方可抑制肿瘤相关巨噬细胞(TAM)浸润和IL-6的分泌。故此提出假说：清胰化积方下调Cyr61调节EMT抑制胰腺癌肝转移作用有赖于肿瘤微环境TAM的参与，Cyr61可能通过调控肿瘤相关巨噬细胞-胰腺癌细胞相互作用反馈环促进胰腺癌细胞的生长和转移。为了验证该假说，我们将采用脂质体载体转染以及胰腺癌细胞和巨噬细胞共培养系统等手段，明确Cyr61调控肿瘤相关巨噬细胞-胰腺癌细胞交互对话促进胰腺癌细胞生长和转移的作用机理；并通过清胰化积方干预研究，进一步阐明中药抗胰腺癌肝转移作用的分子机制。本研究将从Cyr61调控肿瘤相关巨噬细胞-胰腺癌细胞交互对话这个新视点为揭示中药抗胰腺癌肝转移作用机制提供新靶点和新思路。

背景：胰腺癌肝转移预后差，迄今缺乏有效治疗手段，寻找新的治疗靶点将具有重要临床价值。课题组前期面上项目证实清胰化积方下调Cyr61调节EMT抑制胰腺癌肝转移，后续研究中我们又发现清胰化积方可抑制肿瘤相关巨噬细胞(TAM)浸润和IL-6的分泌。故此提出假说：清胰化积方下调Cyr61调节EMT抑制胰腺癌肝转移作用有赖于肿瘤微环境TAM的参与，Cyr61可能通过调控肿瘤相关巨噬细胞-胰腺癌细胞相互作用反馈环促进胰腺癌细胞的生长和转移。.主要研究内容：采用脂质体载体Cyr61过表达手段以及胰腺癌细胞与巨噬细胞共培养系统，通过PANC-1和BxPC-3细胞的研究，定量PCR和ELISA法测定细胞因子如IFN-γ、IL-1、IL-4、IL-6、IL-10、TNF-α、VEGF、TGF-β等的表达和分泌情况, 体内外实验观察对胰腺癌细胞侵袭和迁移能力的影响。清胰化积方干预胰腺癌细胞体外实验，观察对胰腺癌细胞侵袭和迁移能力的影响，Real-time PCR、Western-bolt检测胰腺癌细胞Cyr61、CD31、CD68和CD204表达和EMT相关标志物（E-cadherin、Vimentin等）表达的变化；清胰化积方干预胰腺癌肝转移裸小鼠模型体内实验，观察体内抑瘤效果和肝转移的影响，ELISA法测定细胞因子如IFN-γ、IL-1、IL-4、IL-6、IL-10、TNF-α、VEGF、TGF-β等的表达和分泌情况；Real-time PCR、Western-bolt和免疫组化法检测肝转移组织Cyr61、CD31、CD68和CD204表达和EMT相关标志物（E-cadherin、Vimentin等）表达的变化。.重要结果：Cyr61在胰腺癌组织和细胞中呈高表达，提示患者预后不良。Cyr61促进巨噬细胞的M2极化，增强免疫逃逸，促使胰腺癌细胞EMT，从而促进胰腺癌细胞的生长和转移能力。去泛素酶USP10可抑制YAP1的泛素化降解，促进Cyr61的表达，从而促进胰腺癌细胞中PD-L1和Galenctin9的表达，诱导巨噬细胞M2极化，从而促进胰腺癌细胞的转移和免疫逃逸。清胰化积方下调Cyr61表达，抑制炎性微环境TAM，逆转胰腺癌细胞EMT表达，抑制裸小鼠肝转移。.结论：清胰化积方抗胰腺癌肝转移作用的分子机制是通过下调Cyr61抑制炎性微环境TAM逆转胰腺癌细胞EMT表达。