蝎毒多肽提取物靶向SDF-1/CXCR4信号通道在肝癌化疗期间再增殖治疗机制研究

Residual tumor cells during chemotherapy accelerated repopulation is one of the main reasons for the failure of chemotherapy. Chemotherapy in treatment of liver cancer has side effects and decreases immune function. Chinese medicine treatment has the advantage of overall regulation and has no significant side effects. Adjuvant chemotherapy with Chinese medicine can decrease toxicity, increase efficacy and enhance the role of the immune system. The preliminary studies indicate that the repopulation of tumor cells during chemotherapy in the treatment of H22 tumor model accelerated, and SDF-1/CXCR4 signaling pathways associated with it. On the basis of the preliminary study, we will establish the model of liver cancer repopulation during chemotherapy with the intervention of PESV, and utilize real-time quantitative RT-PCR, immunohistochemistry, western blot analysis, flow cytometry, gene chip to detect the expression of SDF-1 /CXCR4 pathway related cytokines SDF-1, of CXCR4, of ERK1 / 2, FAK, PI3K and analyze the relevance. We will observe the impact of PESV on liver tissue and tumor tissue from whole, tissue, cell, protein, gene level, and screen out target gene with the intervention of PESV, and find out therapeutic targets and molecular mechanisms of liver cancer repopulation during chemotherapy with the intervention of PESV, to provide experimental evidence for Chinese medicine targeting repopulation during the treatment of liver cancer chemotherapy.

化疗期间残存肿瘤细胞加速再增殖是化疗失败的主要原因之一。化疗治疗肝癌副作用大并降低机体免疫功能。中医药治疗具有整体调节优势，无明显毒副作用，辅助化疗能起到减毒增效、增强免疫力的作用。前期研究表明，在H22荷瘤小鼠模型治疗中化疗期间癌细胞再增殖加速，并发现SDF-1/CXCR4信号通路与此相关。我们在前期研究的基础上，拟建立肝癌化疗期间再增殖模型，并用PESV干预，采用实时荧光定量RT-PCR、免疫组织化学、Western blot、流式细胞术、基因芯片等技术检测SDF-1/CXCR4通路中相关细胞因子SDF-1、CXCR4、ERK1/2、FAK、PI3K的表达并分析其相关性。从整体、组织、细胞、蛋白、基因水平观察PESV对肝组织的影响和对肿瘤组织的作用，筛选出PESV干预目的基因， 寻找PESV干预肝癌化疗期间再增殖的治疗靶点及分子机制，为中医药靶向治疗肝癌化疗期间再增殖提供实验依据。

本项目基于SDF-1/CXCR4通路，探讨肝癌化疗期间再增殖与通路中关键分子靶点的相关性，分析传统中药蝎毒多肽提取物（PESV）肝癌化疗期间再增殖治疗机制。本实验成功建立肝癌化疗期间再增殖模型。通过在体和离体实验证实PESV通过下调SDF-1/CXCR4通路及其相关细胞因子对肝癌化疗期间再增殖有明显抑制作用。证实下调CXCR4或SDF-1均可以降低VEGF表达，有效抑制小鼠H22肝癌肿瘤的生长、降低肿瘤组织内的血管生成，同时下调CXCR4和SDF-1对小鼠H22肝癌肿瘤的生长、降低肿瘤组织内的血管生成的影响更显著；证实PESV可以降低PCNA表达和增高survivin表达，抑制了肿瘤细胞增殖和促进了肿瘤细胞凋亡；PESV联合化疗组明显调高CD4+T/CD8+T值，NK细胞明显升高，从而增加了机体免疫力；Real-time PCR及Westernblot检测显示SDF-1/CXCR4及其相关细胞因子FAK 、PI3K和ERK1/2mRNA及蛋白表达下调；表达谱基因芯片研究结果显示，联合组与模型组比较有2238条基因差异表达，其中上调基因1421条，下调基因817条；联合组与化疗组比较有4732条基因差异表达，其中上调基因2864条，下调基因1868条；化疗组与模型组比较有985条基因差异表达，其中上调基因568条，下调基因417条。本实验其基因功能涉及催化活性、代谢过程、免疫反应、应激应答等功能。本课题按项目计划顺利完成。