轴突导向蛋白4D协同血管内皮生长因子促进上皮性卵巢癌血管生成及肿瘤生长的相关研究

Anti-VEGF therapy is still not satisfactory, although it has been regarded as one of the important strategies for advanced ovarian cancer patients. Recently, emerging evidences had certified that semaphorin4D (SEMA4D), protein originally related to control of axonal growth in nervous system development, and its receptor, Plexin-B1, play an essential role in tumor-induced angiogenesis. We had already demonstrated the positive expression of SEMA4D was associated with VEGF closely in epithelial ovarian cancer (EOC) and suggested the shorter progression-free survival and overall survival for the advanced EOC patients. We perform this present study in order to understand SEMA4D inducing EOC angiogenesis better. We prepare human soluble SEMA4D protein and restructure SEMA4D, Plexin-B1, VEGF and VEGFR-2-Short hairpin (sh) RNA. We acquire specific human ovarian cancer cell (SKOV3, A2780 and SW626) and human umbilical vein endothelial cell (HUVEC) for the following experiments through the use of RNA interference and Western blot. Consequently, the expressions of SEMA4D after the blockage of VEGF in ovarian cancer cells are evaluated by immunoblot analysis. The migrations of HUVECs with different treatments are detected by use of Boyden chamber cell migration assay. In vitro and in vivo angiogenesis assays and tumor xenograft experiments, we aim to demonstrate SEMA4D cooperate with VEGF to promote anigogenesis in EOC and can perform the same function in a setting of VEGF blockade. Furthermore, we aim to show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor-resistant tumors, suggesting that his may represent a novel treatment for EOC patients.

靶向VEGF的抗血管生成治疗是晚期卵巢癌的重要治疗策略之一，但疗效并不尽如人意。轴突导向蛋白4D(SEMA4D)是近来备受关注的一类促血管发生分子。前期研究中我们证实上皮性卵巢癌中SEMA4D与VEGF关系密切。为深入了解SEMA4D促卵巢癌血管发生机制，本研究制备SEMA4D可溶蛋白及构建SEMA4D、VEGF、Plexin-B1和VEGFR-2的shRNA载体，分别转染人卵巢癌细胞和人脐静脉内皮细胞（HUVEC）。WB检测抑制卵巢癌细胞中VEGF表达或抗VEGF治疗后SEMA4D的变化。迁移实验观察不同处理HUVEC迁移能力的变化。体内外血管生成实验分析SEMA4D对卵巢癌血管发生的影响，并与VEGF促血管发生作用进行比较。证实SEMA4D促卵巢癌血管生成作用，为晚期卵巢癌患者针对VEGF抗血管生成治疗效果不佳时，联合或补充针对SEMA4D信号途径多靶点抗血管生成治疗的可行性提供依据。

卵巢癌是世界范围内女性生殖器官常见三大恶性肿瘤之一，致死率占各类妇科肿瘤之首。卵巢癌种类繁多，其中上皮性卵巢癌最为常见，占 85-90%。上皮性卵巢癌发病隐匿，70％的女性在确诊为上皮性卵巢癌时已处于晚期发生转移，导致术后复发率高、预后差。因此，研究上皮性卵巢癌晚期生长和转移的分子机制对提高患者的生存率至关重要。肿瘤生长、 侵袭及转移是一个复杂的多步骤过程, 受诸多因素影响。其中，肿瘤的血管生成起着重要作用。. 本研究旨在为晚期上皮性卵巢癌患者抗VEGF抗血管生成治疗效果不佳时，针对SEMA4D/Plexin-B1信号途径多靶点抗血管生成治疗的可行性提供重要的实验和理论依据。本研究发现卵巢癌细胞中SEMA4D与VEGF之间存在相互关联，抑制VEGF的表达后，SEMA4D蛋白表达降低；在上皮性卵巢癌组织中，SEMA4D与VEGF正相关；分别通过shRNA敲降SEMA4D与VEGF的表达后，发现敲降SEMA4D的表达后卵巢癌细胞A2780和人脐静脉内皮细胞HUVEC的迁移和血管生成拟态的能力降低，同时敲降SEMA4D与VEGF细胞的迁移和血管生成拟态的能力明显降低；然后，制备了SEMA4D可溶性蛋白，SEMA4D可溶性蛋白促进A2780和HUVEC的迁移和血管生成拟态的能力；阻断SEMA4D的受体Plexin-B1后可以抵消SEMA4D可溶性蛋白对A2780和HUVEC的迁移和血管生成拟态能力的促进作用; 最后，通过应用小鼠结肠癌细胞CT26（对抗VEGF治疗不敏感细胞系）进行皮下注射成瘤，随后分别或者联合给予anti-VEGF和anti-SEMA4D抗体进行腹腔注射发现联合应用anti-VEGF和anti-SEMA4D抗体后皮下肿瘤的大小较分别注射有明显降低，免疫荧光检测瘤块内血管密度明显降低。该数据说明阻断SEMA4D蛋白表达联合抗VEGF治疗对晚期上皮性卵巢癌患者的治疗有明显疗效。然而，该治疗方案在临床上的疗效仍需要进一步观察。. 本课题在以往研究的基础上，针对临床上皮性卵巢癌患者抗VEGF治疗效果不佳，提出了联合或补充针对SEMA4D信号途径多靶点抗血管生成治疗的可行性依据，为抗VEGF治疗效果不佳的上皮性卵巢癌患者研究提供新的思路，为在抗VEGF治疗效果不佳的上皮性卵巢癌患者治疗中的应用提供证据。