[The establishment of the immortalized mouse brain microvascular pericytes model and its preliminary application in screening of cerebrovascular toxicants].

Objective: To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study. Methods: Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD(50)) was calculated in linear regression. Results: Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD(50) of lead acetate was 2 025.0 μmol/L, the LD(50) of cadmium chloride was 36.6 μmol/L, and the LD(50) of sodium arsenite was 33.2 μmol/L. Conclusion: The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.目的： 建立小鼠脑微血管周细胞永生化模型并应用于脑血管毒物筛查研究。 方法： 取3周龄雄性C57BL/6小鼠2只，取小鼠大脑皮质，用组织分离和酶消化法培养原代小鼠脑微血管周细胞，逆转录病毒转染LT质粒构建永生化细胞株。采用单克隆法纯化细胞，采用细胞免疫荧光鉴定细胞纯度，MTS法绘制细胞生长曲线，将永生化小鼠脑微血管周细胞分别暴露于0、160、320、640、1 280、2 560 μmol/L的醋酸铅和0、5、10、20、40、80 μmol/L的氯化镉及0、5、10、20、40、80 μmol/L的亚砷酸钠24 h后，用MTS法检测细胞活力，用线性回归法计算不同毒物半数致死量（LD(50)）。 结果： 永生化脑微血管周细胞呈长梭形生长，表达周细胞特异性标记物α-平滑肌肌动蛋白（α-SMA），且都表达功能性收缩蛋白中间丝蛋白-波形蛋白（Vimentin）；原代脑微血管周细胞增殖缓慢，接种第3天进入对数生长期，倍增时间为48 h；永生化脑微血管周细胞增殖旺盛，接种第3天进入对数生长期，倍增时间为24 h；永生化脑微血管周细胞生存率随各毒物染毒剂量的增加呈现下降趋势，醋酸铅LD(50)为2 025.0 μmol/L，氯化镉LD(50)为36.6 μmol/L，亚砷酸钠LD(50)为33.2 μmol/L。 结论： 利用逆转录病毒转染LT质粒可成功构建永生化小鼠脑微血管周细胞，并可应用于脑血管毒物筛查研究，对永生化小鼠脑微血管周细胞的毒性大小依次为亚砷酸钠、氯化镉、醋酸铅。.