【作者】 张华蓉；

【导师】 卞修武；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 近年研究发现,在缺血、创伤和肿瘤等病理条件下检测到源于骨髓的内皮前体细胞或称为内皮祖细胞( endothelial progenitor cells, EPCs)迁移进入病变部位增殖、分化并形成新血管,参与组织修复或肿瘤进展。该过程类似于胚胎时期的血管形成或血管发生(vasculogenesis),表明EPCs是成人组织血管新生的重要参与细胞。肿瘤血管新生化在肿瘤的生长和演进中具有重要作用。近来提出的靶向肿瘤血管系统的抗瘤治疗策略不仅针对成熟的内皮细胞,还可能通过抑制EPCs骨髓动员、归巢来抑制血管新生。尽管介导EPCs血管效应的分子机制及EPCs在肿瘤血管新生化过程中募集、归巢、组装成血管过程的确切机制尚不完全明了,但已有研究表明血管内皮生长因子(vascular endothelial growth factor, VEGF)、趋化因子CXCL8 /白细胞介素-8 (interleukin-8, IL-8)、趋化因子CXCL12 /基质细胞衍生因子(stromal cell-derived factor-1,SDF-1)等是EPCs非常重要的调节者,EPCs在肿瘤生长和演进中发挥的重要作用已使其成为抗肿瘤治疗研究的新靶点。我室既往研究证实,自行研制的脂氧化酶抑制剂诺帝(Nordy)不仅具有抑制肿瘤细胞增殖的作用,还具有抗血管生成作用。诺帝能有效抑制分离培养的人脐静脉内皮细胞和人恶性胶质瘤微血管内皮细胞的功能活性,并能减低实验性胶质瘤的微血管密度。但诺帝对EPCs参与肿瘤血管新生过程的影响尚不清楚。本研究以人脐血源性EPCs为材料,研究诺帝对体外培养的EPCs血管形成活性的影响,并采用EPCs参与肿瘤血管新生模型检测诺帝对体内EPCs血管形成效应的影响。从人脐带血中分离培养获得EPCs,在体内、外检测其内皮细胞特性;检测我室自行合成的抗癌化合物诺帝(Nordy)对两个重要的促血管生长因子VEGF和CXCL8介导的EPCs活性的抑制作用,并研究诺帝对U87细胞裸鼠移植瘤中EPCs参与血管新生的影响。主要结果和结论如下:1.分离获得的EPCs具有克隆形成能力,体外诱导培养具有内皮细胞免疫表型(表达CD133、CD34、KDR、vWF和CD31)和功能(吞噬DiI-Ac-LDL,连接FITC-UEA-1,在Matrigel中形成小管样结构);在VEGF刺激下EPCs具有较强的增殖活性、迁移能力和体外形成小管样结构的能力。而100μmol/L诺帝作用24 h明显抑制VEGF诱导的EPCs增殖活性(P<0.05),25～50μmol/L诺帝作用48～72 h也明显抑制EPCs增殖活性(P<0.05);25～100μmol/L诺帝显著抑制VEGF诱导的EPCs迁移活性和体外形成小管样结构的能力(P<0.05)。2. EPCs表达高水平的CXCL8及其受体CXCR1和CXCR2,100μmol/L诺帝作用24 h明显抑制EPCs在蛋白水平(P<0.01)和mRNA水平(P<0.05)表达CXCL8及其受体CXCR1和CXCR2;外源性CXCL8通过刺激活化EPCs表达的CXCL8受体而诱导EPCs迁移运动,100μmol/L诺帝显著抑制CXCL8诱导的EPCs迁移能力(P<0.01)。3. EPCs能特异性归巢到人胶质瘤细胞裸鼠移植瘤组织,并整合形成新生血管。形态上,EPCs来源的内皮细胞与鼠源性内皮细胞相互连接,构成细长条索状的嵌合性微血管;还可见仅由EPCs来源的内皮细胞形成的条索样幼稚血管。EPCs来源的内皮细胞占肿瘤总血管内皮细胞的18%。体内诺帝治疗显著抑制U87移植瘤生长,使其体积减小(P<0.05),微血管密度降低(P<0.05);诺帝明显抑制EPCs整合形成肿瘤新生血管数(P<0.05),并对移植瘤新生血管异质性产生影响,表现为,EPCs来源的内皮细胞形成的幼稚血管结构不明显,管腔样或条索样结构少见,而仅呈单个或几个细胞聚集状态,与鼠源性内皮细胞连接不紧密。诺帝显著抑制移植瘤组织的VEGF和CXCL8蛋白表达(P<0.05)。结论:EPCs具有形成血管能力,并参与异种移植瘤血管新生,提示EPCs在恶性肿瘤血管新生过程中具有重要作用。体内、外研究表明诺帝可能通过抑制VEGF和CXCL8诱导的EPCs活性进而减少EPCs整合形成新生血管并影响新生血管异质性而发挥其抗肿瘤效应。更多还原

【Abstract】 Both angiogenesis and vasculogenesis contribute to tumor neovascularization, which provides oxygen and nutrients for tumor cell survival and proliferation. Endothelial progenitor cells (EPCs) are important initiators of vasculogenesis in the process of tumor neovascularization. Accumulating evidences indicate that EPCs are actively recruited to sites of tumor where the cells differentiate in situ into mature ECs and integrate into new blood vessels. However, it is unclear how circulating EPCs contribute to the formation of tumor microvessels. EPCs can be mobilized from bone marrow into the blood stream and homed by a variety of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and chemokines such as CXCL12 (stromal derived factor-1, SDF-1) and CXCL8 (interleukin-8, IL-8). Therefore, EPCs have been considerated as a promising target in anti-angiogenesis treatment strategy.Nordy is a chiral compound of a natural lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), synthesized in our laboratory. In previous study, we found that Nordy inhibited glioma cells proliferation, induced glioma cell apoptosis. Furthermore, it has been confirmed that Nordy inhibited angiogenesis in inflammatory diseases and cancers through its inhibitory effect on CXCR4-mediated production of CXCL8 and VEGF. It has also been found that Nordy could inhibit migration of glioma-derived microvascular endothelial cells to form tubule-like structures. However, the effect of Nordy on EPCs has not been identified.To investigate whether Nordy has influence on vasculogenic function of EPCs and their contribution to malignant glioma neovascularization, we isolated and cultured EPCs from human umbilical cord blood (HUCB) and examined their morphology and immunophenotype. Then, we explored the inhibitory effect of Nordy on the the response of EPCs to VEGF and CXCL8. Finally, we studied the influences of Nordy on EPCs contributions to malignant glioma neovascularization. The main results and conclusions are as follows:1. We isolated EPCs from HUCB. These EPCs expressed CD133, CD34, VEGFR-2 (KDR), CD31 and von Willebrand factor (vWF), and were capable of forming colonies. They could functionally uptake acetylated low-density lipoprotein (ac-LDL) and bind lectins. These EPCs were highly proliferative, migrated and formed capillary-like tubules in response to VEGF. Incubation of EPCs with 100μmol/L Nordy for 24 h initially inhibited the proliferative capacity of EPCs induced by VEGF (P < 0.05). Moreover,25～50μmol/L Nordy also exhibited inhibitory effect at 48～72 h. In addition, 25～100μmol/L Nordy impaired EPCs migratory and tubule-forming capability in vitro (P < 0.05).2. EPCs expressed high levels of CXCL8, CXCR1 and CXCR2 at mRNA and proteins, but reduced by treatment with 100μmol/L Nordy. Moreover, 100μmol/L Nordy impaired EPCs migratory capacity in vitro induced by CXCL8 (P < 0.05).3. When injected into mice bearing subcutaneously implanted human malignant glioma, EPCs specifically accumulated at the sites of tumors and differentiated into mature endothelial cells (ECs), which formed 18% of the tumor microvessel ECs. Nordy inhibited the growth of glioma xenografts, reduced tumor volume and microvessel density. Also, treatment with Nordy significantly inhibited production of VEGF and CXCL8 in tumor and attenuated incorporation of EPCs into tumor neovascularture. In addition, Nordy obviously influenced the morphology of tumor microvessles,that is, Nordy weakened the capability of EPCs-derived ECs to form typical lumina or strap-like structures in tumor, but EPCs-derived ECs just arranged by sing cell or clusters.In summary, the present results suggest that (1) EPCs constitute important components of tumor microvessel network and contribute to tumor neovascularization. (2) Nordy inhibited EPCs function induced by VEGF or CXCL8 in vitro and attenuated incorporation of EPCs into tumor neovascularture and also impacted tumor microvascular phenotype heterogeneity. This might be one of the mechanisms of the anti-cancer effection of Nordy.更多还原