低氧对体外培养的血管平滑肌细胞HMGB1的表达及尼莫地平干预作用的研究-神经病学专业论文.docx

华中科技大学硕士学位论文 华 中 科 技 大 学 硕 士 学 位 论 文 PAGE 1 PAGE 1 低氧对体外培养的血管平滑肌细胞 HMGB1 的表达及 尼莫地平干预作用的研究 华中科技大学同济医学院附属协和医院神经内科 硕士研究生：陈光辉 导 师：刘昌勤 中文摘要 目的：探讨低氧对血管平滑肌细胞分泌高迁移率族蛋白 B1（high mobility group protein box1，HMGB1）的影响，以及尼莫地平对其干预作用。 方法：体外培养大鼠血管平滑肌细胞，采用 Kuwabara 等并加以改进的方法对平滑肌 细胞进行低氧培养，在低氧处理后（1h、4h、12h、24h 和 48h），使用 Western blot 检 测培养液上清中 HMGB1 的表达水平。再按同样方法加入尼莫地平预处理，分为对照 组、单纯低氧组、低氧+尼莫地平组（加入 100mmol/L 尼莫地平），低氧培养 12h 后检 测上清 HMGB1 水平。台盘兰排斥法检测 48h 以内细胞的存活率。 结果：与正常对照组相比，培养液上清中 HMGB1 在低氧各组表达均明显增加 （P0.05）。低氧组、缺氧+尼莫地平组 HMGB1 表达水平明显高于对照组，尼莫地平 处理组较单纯缺氧组要低（P0.05）。台盼蓝排斥法检测低氧 48h 以内对细胞生存率 影响较小。 结论：低氧可诱导体外培养的血管平滑肌细胞表达分泌 HMGB1，在 24h 内随着缺氧 时间的延长，其表达增加。加入尼莫地平预处理可降低 HMGB1 的表达,提示尼莫地平 可能通过降低 HMGB1 水平从而起到脑保护作用。 关键词： 低氧 血管平滑肌细胞 HMGB1 尼莫地平 A study on protein expression of HMGB1 in cultured vascular smooth muscle cells (VSMCs) under hypoxia and the interventional effect of Nimodipine Department of neurology of Union Hospital, Tongji Medical College huazhong University of Science Technology Master Candidate: Chen Guanghui Supervisor: Prof. Liu Changqin ABSTRACT Objective： To observe the effects of hypoxia on the expression of HMGB1 in cultured VSMCs. Method: The cultured VSMCs of rats were divided into six groups and they were cultured in normal condition and under hypoxic condition for 1, 4, 12, 24 and 48 hours. The levels of HMGB1 in the supernatant were measured by Western blot. Subsequently, we administrated nimodipine to pretreatment. The cells were divided into three groups: normal controlled group，hypoxic group and nimodipine-pretreated hypoxic group. HMGB1 were measured after 12 hours’ hypoxic culture. The cell viability within 48h was examined by trypan blue exclusion test. Results: Compared with normal control, the expression of HMGB1 in the hypoxic cultured supernatant was significantly increased （ P0.05 ） . But the HMGB1 level in nimodipine-pretreated hypoxic group was lower than the hypoxic group（P0.05）.Within 48h, no differences in cell viability were found compared with control group by