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目的:探讨胶质瘤U87细胞培养上清诱导CD133+内皮细胞血管生成及其可能的机制。方法:磁珠法分选脐血CD133+细胞,体外诱导为CD133+内皮细胞并以共聚焦显微镜加以鉴定。U87细胞培养上清作用于CD133+内皮细胞(以DMEM作用为对照),体外血管生成实验检测其体外血管生成,Transwell法检测其迁移能力,Western blotting检测CD133+内皮细胞中VEGFR-1、VEGFR-2与基质金属蛋白酶9(matrix metalloproteinase,MMP-9)的表达,明胶酶谱检测MMP-9的活性。结果:脐血CD133+细胞体外诱导培养14d后,约90%的细胞呈Dil-ac-LDL和FITC-UEA-1双阳性,具有内皮细胞特性,鉴定为CD133+内皮细胞。与DMEM对照组相比,U87细胞培养上清可诱导CD133+内皮细胞血管生成[(40.7±3.3)vs(21.0±2.3),P<0.05],促进CD133+内皮细胞迁移[(0.60±0.04)vs(0.27±0.02),P<0.05]。U87细胞培养上清上调CD133+内皮细胞中VEGFR-2与MMP-9的表达(P<0.05),而VEGFR-1的表达基本不变;U87细胞培养上清还可增强CD133+内皮细胞中MMP-9的酶活性。结论:胶质瘤U87细胞培养上清通过上调CD133+内皮细胞中VEGFR-2与MMP-9的表达促进内皮细胞血管生成。
Objective: To investigate whether glioma U87 cell culture supernatant induces CD133 + endothelial cell angiogenesis and its possible mechanism. Methods: The cord blood CD133 + cells were sorted by magnetic beads method, induced by CD133 + endothelial cells in vitro and identified by confocal microscopy. U87 cell culture supernatant was used to act on CD133 + endothelial cells (with DMEM as control). The in vitro angiogenesis assay was used to detect the angiogenesis in vitro. Transwell assay was used to detect the migration ability of the cells. Western blotting was used to detect the expressions of VEGFR-1, VEGFR- Matrix metalloproteinase 9 (MMP-9) expression, gelatin zymography MMP-9 activity. Results: After cultured for 14 days, about 90% of cord blood CD133 + cells were double positive for Dil-ac-LDL and FITC-UEA-1, with endothelial cell characteristics and were identified as CD133 + endothelial cells. Compared with DMEM control group, U87 cell culture supernatant could induce angiogenesis of CD133 + endothelial cells [(40.7 ± 3.3) vs (21.0 ± 2.3), P <0.05], promote the migration of CD133 + endothelial cells [(0.60 ± 0.04) vs 0.27 ± 0.02), P <0.05]. U87 cell culture supernatant upregulated the expression of VEGFR-2 and MMP-9 in CD133 + endothelial cells (P <0.05), while the expression of VEGFR-1 remained unchanged. U87 cell culture supernatant also enhanced the expression of MMP-9 in CD133 + endothelial cells Enzyme activity. Conclusion: The glioma U87 cell culture supernatant can promote the angiogenesis of endothelial cells by up-regulating the expression of VEGFR-2 and MMP-9 in CD133 + endothelial cells.