VEGF对大鼠星形胶质细胞的缺氧保护作用及其机制

目的 观察缺氧对体外培养的大鼠星形胶质细胞的影响，探讨外源性血管内皮细胞生长因子（VEGF）对大鼠星形胶质细胞缺氧的保护作用及其可能的机制。方法 体外原代培养大鼠脊髓星形胶质细胞并鉴定（胶质原纤维酸性蛋白GFAP阳性）。星形胶质细胞分别经不同时间的缺氧处理、缺氧处理前加入不同浓度（10～100ng／ml）VEGF处理以及同时加入VEGF受体Flk一1阻断剂SU1498处理后，应用光镜、细胞计数、MTT法、TUNEL标记及免疫组化技术检测星形胶质细胞形态、细胞增殖活性和凋亡、VEGF和Flk-1的表达改变。结果 纯化培养1周的细胞，经鉴定其星形胶质细胞纯度达98％；8～12h缺氧可明显诱导星形胶质细胞活化，表现为细胞胞体变大、胞突变粗、GFAP表达升高，细胞增殖活性降低、凋亡指数增加、VEGF和Flk-1表达增加；缺氧处理前20～24h加入50ng／ml VEGF可使细胞活力明显增强，细胞凋亡指数明显降低，细胞缺氧损伤明显改善；而700ng／ml SU1498可明显阻断或抑制VEGF对星型胶质细胞的缺氧保护作用。结论 缺氧可诱导大鼠星形胶质细胞反应性活化，外源性VEGF可能通过VEGF受体Flk-1发挥对大鼠星形胶质细胞的缺氧保护作用。

Neuroprotective Effect of Exogenous Vascular Endothelial Growth Factor on Anoxic Rat Spinal Cord Astrocyte and the Underlying Mechanism

Objective To observe the effect of hypoxia on primary cultured rat spinal cord astrocyte and investigate the neuroprotective effect of exogenous vascular endothelial growth factor(VEGF) on anoxic astrocyte in vitro.Methods The rat embryonic spinal cord astrocytes were cultured and identified by cellular morphology and glial fibrillary acidic protein(GFAP).The astrocytes were treated with VEGF at different concentrations(10 ng/ml-100 ng/ml) as well as with VEGF receptor(Flk-1) inhibitor SU1498.Then the astrocytes were exposed to hypoxia for different time.The light microscope,MTT assay,TUNEL labeling and SABC immuno-histochemistry were used to measure and observe the changes of the astrocytes in cell morphology,growth,proliferation,apoptosis,VEGF and Flk-1 expression.Results GFAP identified that 98% of the purified 1-week cultured cells were astrocytes.8-12 hours hypoxia obviously induced the activity of astrocyte.The cytobody became bigger,the cytoskeleton grew thicker.The activity of proliferation decreased but apoptosis index increased.The GFAP,VEGF and Flk-1 expression increased.The treatment with 50 ng/ml VEGF 20-24 hours before hypoxia apparently enhanced the activity,decreased the apoptosis index of the astrocytes,and hence improved the hypoxia-injuried astrocytes.Howerver,700 ng/ml SU1498 obviously inhibited the neuroprotective effect of VEGF on anoxic astrocytes.Conclusion Hypoxia induces reactive activity of astrocyte.The exogenous VEGF exerts neuroprotective effect on astrocytes via VEGF receptor-Flk-1.