蜂毒素与组织蛋白酶S对肝癌血管生成调控机制研究

In previous studies we have shown that Cathepsin S (Cat S) plays an important role in tumor formation, vascular invasion and metastasis processes. Evidence was found that Cat S is also influencing the invasion and metastasis of heptacellular carcinoma (HCC). The studies indicate that Melittin inhibits angiogenesis in HCC significantly, hence the question arises whether Mellitin can be regulating the tumor angiogenesis via the Cat S molecule? We are not aware of any conclusive literature on this question. It was previously shown that Melittin and Cat S could control vascular endothelial growth factor (VEGF). The latter can be formed through the VEGF-ERK/MAPK pathway which influences angiogenesis of HCC. This would support a hypothesis that Mellitin through the Cat S VEGF-ERK/MAPK pathway regulates the angiogenesis of the tumor of HCC. To test this hypothesis, we intend to use ShRNA Cat S transfection with a MHCC97-H cell line which has been selected for high expression of Cat S in human HCC. An in vitro and in vivo model will be employed for the study of the Melittin influence on the growth of downstream proteins in MHCC97-H for carcinoma angiogenesis. Application of protein chip, co- immunoprecipitation and another techniques will determine a possible influence of Melittin targeting Cat S in the VEGF-ERK/MAPK pathway and thus regulating the mechanism of HCC. This study will lay the foundation for the formation mechanism from Cat S ，which new perspective for revealing of melittin on HCC.

组织蛋白酶S（CatS）在肿瘤血管形成、侵袭转移中起重要作用。研究表明蜂毒素具有抑制肝癌血管生成作用，但蜂毒素是否通过CatS调控肿瘤血管形成？迄今未见报道。我们前期研究发现CatS与肝癌侵袭转移特性相关；基于蜂毒素和CatS均可调控VEGF，而后者可通过VEGF-ERK/MAPK通路参与肿瘤血管形成，因此提出蜂毒素是否通过CatS调节VEGF-ERK/MAPK 通路影响肝癌血管形成假说。为验证此假说，拟应用ShRNA Cat S转染已筛选出的高表达CatS的MHCC97-H人肝癌细胞系高表达株，通过体内外实验观察蜂毒素对MHCC97-H生长影响；再采用蛋白芯片、免疫共沉淀等技术探索蜂毒素调节VEGF-ERK/MAPK通路的靶蛋白，进一步阐明蜂毒素与CatS抑制肝癌血管生成的作用机制。本项目将从CatS这个新视点出发，为揭示蜂毒素抗肝癌血管生成机制奠定理论基础。

本课题组前期研究已经证实蜂毒素能抑制HCC细胞增殖和移植瘤生长等作用。这提示血管内皮细胞在形成血管的几个关键环节可能和蜂毒素抑制作用有关。另外，在MHCC97-H细胞沉默Cat S基因显着抑制血管内皮生长因子分泌和抑制人脐静脉内皮细胞管状结构形成。根据以上结果我们推测，蜂毒素靶向作用于Cat S，具有抗血管生成、抑制肿瘤转移的重要作用，为此我们通过研究沉默Cat S是否影响MHCC97-H细胞的生物学功能，并进一步探讨蜂毒素是否通过作用Cat S抑制肿瘤生长和肝癌血管行成;同时揭示在蜂毒素作用下，Cat S与VEGF-A/VEGFR2/MEK1/ERK1/2相关通路中关键蛋白分子间相互调节作用否影响肝癌血管的形成的机制;明确沉默Cat S明显影响MHCC97-H细胞的生物学行为;蜂毒素可能通过调控Cat S，抑制肿瘤生长和肝癌血管生成，提示蜂毒素可能是Cat S的抑制剂，有望成为一种潜在的治疗肝癌药物。