STUDY ON MECHANISM OF TUMOR ANGIOGENESIS IN UROTHELIAL CANCERS AND SCREENING OF ANGIOGENIC INHIBITORS

肿瘤血管生成是肿瘤生长和转移所必需的。实体肿瘤依靠血管生成来生长，直径超过几毫米。为探讨血管生成因子在尿路上皮癌血管生成中的作用，我们检测了这些血管生成因子在膀胱癌和肾癌组织中的表达和定位。此外，在三维共培养实验系统中还检测了血管内皮细胞生长因子和b-成纤维细胞生长因子对与肾癌细胞共培养的微血管内皮细胞的微管形成的影响。逆转录聚合酶链式反应检测肾癌组织中b-FGFmRNA、VEGF、PDGF-A和转化生长因子-α的表达分别为96%、77%、69%和50%，膀胱癌中分别为77%、62%、62%和0%。免疫组织化学分析表明，b-FGF和VEGF主要定位于胞核和胞浆。在三维共培养实验系统中，外源性b-FGF和VEGF以剂量依赖的方式增加微血管内皮细胞的管状形成。而抗b-FGF(10µg/ml)和抗VEGF(10µg/ml)抗体可完全抑制系统中血管的形成，提示抗血管生成因子的中性抗体可能通过抑制肿瘤血管生成而抑制肿瘤生长。最近，我们观察到在荷有膀胱癌的裸鼠中，转化生长因子-α的表达随着肿瘤大小的增加而增加，尽管在原始的膀胱癌中转化生长因子-α的表达很弱。此外，我们还观察到，高表达PD-ECGF的膀胱肿瘤表现出由von-Willebrand因子决定的高肿瘤微血管密度。提示多种血管生成因子参与了尿路上皮癌肿瘤血管生成的多步骤过程。针对这些血管生成因子的特异性抗体可能在临床上应用于癌症的治疗。

Tumor angiogenesis is essential for tumor growth and metastasis. Solid tumors depend on angiogenesis to grow larger than a few millimeters in diameter. To investigate the role of several angiogenic factors, such as b-FGF,VEGF,PDGF-A and TGF-alpha in angiogenesis in urothelial cancers, we examined the expression and localization of these angiogenic factors in patients with bladder cancers and renal cell carcinomas. Furthermore, tubulogenesis by VEGF and b-FGF of microvascular endothelial cells co-cultured with renal cancer cells was also examined by a three dimensional co-culture assay system. RT-PCR analysis detected mRNAs of b-FGF,VEGF,PDGF-A and TGF-alpha in 96%, 77%, 69% and 50% in renal cancers and 77%, 62%, 62% and 0% in bladder cancers, respectively. Immunohistochemical analysis revealed that b-FGF and VEGF were primarily localized in the nucleus and cytosol, respectively. Exogenous addition of b-FGF and VEGF increased tube formation of microvascular endothelial cells in a dose dependent manner in the three dimensional co-culture assay system. However, specific antibodies against b-FGF (10mug/ml) and VEGF (10mug/ml) completely inhibited the tube formation in the system, suggesting that neutral antibodies against angiogenic factors, including b-FGF and VEGF may suppress tumor growth by the inhibition of tumor angiogenesis. Recently, we observed that the expression of TGF-alpha increased as a function of tumor size in nude mice bearing bladder tumors, although the expression of TGF-alpha in the original bladder tumor was weak. Furthermore, we observed that bladder tumors which highly express PD-ECGF showed high tumor microvascular density determined by von-Willebrand factor. Thses results suggest that several angiogenic factors were involved in the multi-steps of tumor angiogenesis of urothelial cancers. Specific antibodies against these angiogenic factors may be clinically applicable to the cancer treatment.