【作者】 张弢； 关明； 吕元；

【Author】 Zhang Tao Guan Ming Lu-Yuan Center of Laboratory Medicin, Huanshan Hospital of Fudan University, shanghai 200040,China

【机构】 复旦大学华山医院检验医学中心；

【摘要】 目的:脑胶质瘤源自脑内神经胶质细胞,是最常见的脑部恶性肿瘤,其恶性表型以迅速增殖、局部侵袭和血管生成为主要特征。鉴于色素上皮细胞衍生因子(PEDF)的神经营养、神经保护特性,“胶质细胞静息”效应和对新生血管的抑制作用,本研究评价重组色素上皮细胞衍生因子 (rPEDF)对与胶质瘤有关的血管活性因子、增殖和侵袭的影响。方法:酶联免疫吸附实验(ELISA)测定血管内皮细胞生长因子(VEGF)与血小板反应素-1(17SP—1)的变化; MTT[四甲基偶氮唑蓝,3-(4,5-dimthy-2-2 thiazoly)法检测胶质瘤细胞增殖率;细胞侵袭实验检测rPEDF抑制胶质瘤细胞侵袭性。建立SCID小鼠模型, rPEDF对胶质瘤进行治疗,免疫组化检测C D 3 4,并计算抑瘤率。参照 Weidner方法计数肿瘤内微血管密度(Microvascular density, MVD)。结果:实验数据显示:rPEDF在下调VEGF的表达的同时明显增加TSP-1的表达(P<0．001),显示出抑制胶质瘤血管新生的特性。当rPEDF浓度达时25ng／ml时, 就开始抑制胶质瘤细胞的增殖;当其作用浓度增加时,抑制率也增加,两者之间存在着明显的量效关系;侵袭实验结果都表明,将rPEDF加入胶质瘤细胞后, A172细胞穿透数目有明显下降,即使在加入促进胶质瘤血管生成最有效因子的 -VEGF的条件下,肿瘤细胞穿透数仍明显低于对照组,差异具有显著性(P<0．05),表明rPEDF能有效抑制胶质瘤细胞的体外侵袭性,甚至在VEGF存在的条件下。本研究采用皮下接种法首先成功的建立了裸鼠的胶质瘤模型,实验组在接受rPEDF瘤周注射后,瘤块生长速度明显慢于只接受 PBS注射的对照组,抑瘤率为53％,表明rPEDF可以抑制胶质瘤的生长。已经有报道指出,虽然PEDF是一个多功能的蛋白,它抑制肿瘤生长主要依赖的是它的抗血管生成的功能。我们通过检测微血管密度来评价rPEDF对胶质瘤血管新生的影响。免疫组化结果表明对照组小鼠CD34染色强阳性,提示胶质瘤中有丰富的新生血管的存在;rPEDF处理组小鼠可见明显的微血管密度减低,定量分析结果表明微血管密度减少达55％,这与rPEDF处理组肿瘤体积减少53％的结果相近,从而提示胶质瘤血管新生的减少是导致肿瘤生长缓慢的机制之一。更多还原

【Abstract】 Objective: Gliomas are the most frequent tumors in the central nervous system accounting for about 50% all primary brain tumors. In view of the neurotrophic, anti-angiogenic and "gliastatic" effect of PEDF, we considered that it acts as a potential therapeutic agent against diseases of the central nervous system, especially gliomas. To assess the impact of recombinant pigment epithelium-derived factor( rPEDF) on the growth and invasion of the glioma cells. Methods: The changes of vascular enthothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) were evaluated by enzyme-linked immunoadsorbent assay (ELISA), cell proliferation assays were performed by MTT assay, the cell migratory assay is used for evaluating its inhibitory migration rate. We conducted successfully the subcutaneous xenotranspiantation of glioma in severe combined immu-nodeficient mice. We determined microvessel density (MVD) to assess the impact of treatment on angiogenesis. Results: The rPEDF inhibited the proliferation of glioma cells through increasing the VEGF as well as decreasing the TSP-1( P <0.001), rPEDF could decrease the migratory capacity of the glioma cells even in the presence of VEGF. We determined microvessel density (MVD) to assess the impact of treatment on angiogenesis. Tumors from animals receiving PBS showed intense staining for CD34, indicating the presence of extensive angiogenesis in this type of tumor. Tumors from rPEDF-treated animals showed a marked reduction in MVD. Quantitative analysis demonstrated 55% reduction in the intratumoral microvessel density in rPEDF-treated animals. A 55 % decreased MVD in the PEDF-treated glioma closely paralleled the 53% decreased tumor size inferring a direct relationship between vascularity and growth of gliomas. These observations confirmed the theory that blocking the vascular supply to a tumor significantly curbs its ability to grow.更多还原