【摘要】 目的探索血管内皮生长因子(VEGF)对兔肾血管内皮细胞(MVECs)体外培养的影响。方法采用三步梯度筛网法,获得纯度较高的肾血管球,0.5%胶原酶Ⅳ37℃消化15～20min,离心沉淀获取血管内皮细胞,分加与不加VEGF培养。对培养的细胞用倒置显微镜观察常规形态,用免疫组化法进行第Ⅷ因子相关抗原鉴定,用透射电镜观察细胞浆WeibelPalade小体,用间接免疫荧光法检测CD31、CD34及CD44的表达。结果加VEGF的MVECs原代3d铺满瓶底,3～4d传一代;不加VEGF的MVECs原代7～12d铺满瓶底,7～8d传一代。鉴定时两种培养的MVECs表达是一致的。倒置显微镜观察细胞铺满瓶底时呈典型的铺路卵石样结构,免疫组化法第Ⅷ因子相关抗原阳性,透射电镜观察到细胞浆中的WeibelPalade小体,间接免疫荧光法检测CD31、CD34表达阳性,CD44阴性。结论VEGF对MVECs体外培养的影响无显著意义。更多还原

【Abstract】 Objective To study the role of vascular endothelial growth factor(VEGF) on the renal microvascular endothelial cells(MVESs) in vitro culture.Methods The highly pure glomerruli were harvested by three-step grade filtrate net,followed by digestion with 0.5% Ⅳ collagenase at 37℃ for 15-20min with the transplant methods of isolated glomerali.MVECs were cultured in vitro with or without VEGF,and identified by immunohistochemical SP methods and observed under transmission electron microscope.The antigen CD31,CD34,CD44 were detected by indirect immunofluorescence methods.Results The culture of MVECs adding VEGF formed a generation in 3-4 days,and not adding VEGF in 7-8 days.The highly pure MVECs were cultured in vitro and maintained 18 to 23 generations,and factor Ⅷ-related antigen was positive by immunohistochemical SP method,and Weibel-Palade bodies were observed under transmission electron microscope.It was showed that CD31 and CD34 were positive,and CD44 was negative with indirect immunofluorescence methods.Conclusion VEGF is not a necessity for MVECs culture in vitro.更多还原