大鼠脑微血管周细胞的分离和鉴定

Isolation and identification brain microvessel pericytes in rats

目的探讨大鼠脑微血管周细胞的分离、培养和鉴定方法。方法10只3周龄Wistar大鼠，无菌分离脑组织，采用2次酶消化和1次密度梯度离心法分离脑微血管片段，接种于35mm培养皿进行原代培养。采用相差显微镜观察细胞形态，免疫荧光法鉴定α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、神经元-胶质抗原2（neuron—glial antigen2，NG2）、von Winebrand因子（von Willebrand factor，vWF）和胶质纤维酸性蛋白（giial fibrillary acidic protein，GFAP）相关抗原，甲基噻唑基四唑法测定细胞生长曲线。结果周细胞从贴壁的脑微血管片段周围爬出，呈多角性，12～14d后细胞融合95％。免疫荧光染色显示，周细胞分子标志物仪一SMA和NG2相关抗原呈双阳性，vWF和GFAP相关抗原呈双阴性，证实培养细胞为脑微血管周细胞。原代细胞初始生长速度较慢，传代细胞36～60h进入对数生长期，72～108h进入平台期。结论该方法能成功分离出纯度较高的大鼠脑微血管周细胞。

Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immuno- fluorescence assay was used to identify the associated antigens, such as the a-smooth muscle actin （α-SMA）, neuron-glial antiggen 2 （NG2）, yon Willebrand factor （vWF）, and glial fibrillary acidic protein （GFAP）. Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragnents around, showing polygonal, and the cell fusion was 95% after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens as showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.