Heparanase基因介导宫颈癌淋巴管生成的分子机制研究

The most common way of metastasis of cervical cancer is lymphatic metastasis. Moreover, lymphangiogenesis is an important event in process of tumor metastasis. Therefore, molecular mechanisms of lymphangiogenesis in cervical cancer are attracting more and more attention. We previously showed that heparanase correlates closely with lymphangiogenesis of cervical cancer,and VEGF-C expression mediates by heparanase. Using RNA interference and lipofectamine transfection, we will up-regulate and down-regulate the expression of heparanase, and monitor activity of PI3K/Akt/mTOR and COX-2/PGE2/ERK signaling pathways,then observe expression levels of VEGF-C. We also explore effects of heparanase on lymphatic endothelial cells by three-dimensional lymphangiogenesis assay. Using CHIP technology, we explore target genes interact with heparanase and molecular mechanisms of lymphangiogenesis in cervical cancer. This subject mainly studies heparanase dynamically in the cells, animals and clinical specimens, clarifies the relationship between heparanase and the progression and lymphangiogenesis of cervical cancer.Our study will provide new target for regulating lymphangiogenesis and inhibiting lymphatic metastasis of cervical cancer.

淋巴道转移是宫颈癌最常见的转移途径，而淋巴管生成是肿瘤发生淋巴道转移过程中的重要事件。因此，宫颈癌淋巴管生成机制的研究日益引起重视。申请者前期发现，Heparanase与宫颈癌淋巴管生成密切相关，且Heparanase可介导VEGF-C表达。本课题拟采用RNA干扰和脂质体转染技术，分别上调和下调宫颈癌细胞Heparanase表达，监测PI3K/Akt/mTOR及COX-2/PGE2/ERK通路活性和对VEGF-C表达的影响；利用三维淋巴管形成实验检测Heparanase对淋巴管内皮细胞的作用；采用CHIP技术探寻与Heparanase相互作用的靶基因，探讨Heparanase促进宫颈癌淋巴管生成的分子机制。本课题拟在细胞、动物及临床标本中对Heparanase进行动态研究，揭示Heparanase与宫颈癌发生发展和淋巴管生成的关系，为调控宫颈癌淋巴管形成和抑制宫颈癌淋巴道转移提供新靶点。

淋巴管形成是肿瘤发生淋巴道转移的结构基础。研究发现，Heparanase与肿瘤淋巴管形成密切相关，且Heparanase可介导VEGF-C表达。但Heparanase调控宫颈癌淋巴管形成的具体分子机制尚需进一步研究。本研究检测80例临床肿瘤标本中Heparanase，COX-2，HIF-1a, VEGF-C 和D2-40表达情况，统计学分析它们之间的相关性； 将构建的含Heparanase cDNA的质粒转染宫颈癌HeLa和SiHa细胞24小时，在常氧和低氧环境下培养48小时，检测Heparanase，HIF-1α, VEGF-C表达情况；针对Heparanase设计SiRNA，分别转染宫颈癌HeLa和SiHa细胞24小时，在常氧和低氧环境下培养48小时，然后检测Heparanase，HIF-1α,VEGF-C表达情况；将构建好的含Heparanase cDNA的质粒瞬时转染至HeLa和SiHa细胞，并与人淋巴管内皮细胞（LEC）共培养，检测淋巴管形成能力的变化； 建立裸鼠移植瘤模型，再检测相关指标。.本研究发现：Heparanase 与COX-2，HIF-1α和VEGF-C 表达成正相关，Heparanase高表达的病例淋巴管数量多；低氧环境下，宫颈癌HeLa和SiHa细胞中Heparanase表达水平增强；转染Heparanase cDNA后，HIF-1α和VEGF-C表达增高；宫颈癌和人淋巴管内皮细胞混合培养发现，Heparanase可促进淋巴管形成；体内实验发现，Heparanase高表达可促进肿瘤生长和肿瘤淋巴管形成。本研究揭示了Heparanase可促进宫颈癌淋巴管生成及相关的分子机制，为调控宫颈癌淋巴管形成和抑制宫颈癌淋巴道转移提供实验依据。