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背景:星形胶质细胞是中枢神经系统中分布最广泛的细胞之一,但其在中枢神经退行性疾病中的作用目前尚未明确。目的:观察脂多糖对星形胶质细胞内钙离子、活性氧、一氧化氮水平和细胞存活率的影响。设计:观察对比实验。单位:苏州大学附属第二医院脑循环研究室。材料:实验于2005-05/2005-08在苏州大学附属第二医院脑循环研究室完成,选择8只新生1～3d内SD乳鼠,雌雄不限,由苏州大学动物中心提供,[许可证号:SYXK(苏)2002-0037];脂多糖(Sigma公司),MTT和一氧化氮测定试剂盒(碧云天生物技术有限公司)。方法:参照KevinSt.McNaught等人的方法体外分离培养新生大鼠皮质星形胶质细胞,经反复传代培养和纯化后,根据加入脂多糖剂量的不同将星形胶质细胞分为5组:空白对照组、脂多糖5mg/L组、脂多糖10mg/L组、脂多糖20mg/L组和脂多糖40mg/L组,脂多糖对星形胶质细胞的作用时间为30min和60min。MTT法测脂多糖作用细胞30min和60min细胞存活率;Griess法测量脂多糖作用细胞30min上清液一氧化氮的含量;采用激光共聚焦显微镜观察不同剂量脂多糖作用细胞30min对离体大鼠星形胶质细胞内钙离子、作用细胞60min时活性氧水平的影响,采用相对荧光强度百分比表示星形胶质细胞内钙离子浓度的变化及细胞释放活性氧的水平。主要观察指标:各组星形胶质细胞存活率;星形胶质细胞上清液中一氧化氮的含量;激光共聚焦显微镜下星形胶质细胞内游离钙和活性氧水平的变化。结果:①脂多糖作用星形胶质细胞60min时,脂多糖5mg/L,10mg/L,20mg/L组的细胞存活率均升高,与对照组相比差异均有显著性(P<0.05),脂多糖10mg/L组的细胞存活率高于脂多糖5mg/L组和脂多糖20mg/L组。②脂多糖40mg/L作用细胞30min后引起一氧化氮水平的升高,与对照组和其余3个剂量组相比差异有显著性(P<0.05)。③脂多糖5mg/L,10mg/L,20mg/L组细胞内游离钙的相对荧光强度是其相应的基础状态的200～400倍,脂多糖40mg/L组的荧光强度是其相应的基础状态的1000倍作用;④脂多糖5mg/L,10mg/L组,20mg/L组的活性氧水平是其基础状态的200～300倍,而脂多糖40mg/L组的绝对荧光强度峰超过了检测的范围,推测脂多糖40mg/L组的活性氧水平至少是相应基础状态的300～400倍。结论:脂多糖作用于星形胶质细胞后可使其胞钙离子浓度升高,活性氧和一氧化氮释放增加,促进细胞增殖。
BACKGROUND: Astrocytes are one of the most widely distributed cells in the central nervous system, but their role in CNS degenerative diseases is not yet known. Objective: To observe the effects of lipopolysaccharide on intracellular calcium, reactive oxygen species, nitric oxide and cell viability in astrocytes. Design: observe the contrast experiment. Unit: Second Affiliated Hospital of Soochow University, Department of Brain Circulation. MATERIALS: The experiment was performed at the Brain Circulation Laboratory of the Second Affiliated Hospital of Soochow University between May 2005 and August 2005. Eight SD neonatal SD rats, 1 to 3 days old, were selected, both male and female, and provided by the Animal Center of Soochow University. [License SYXK (Su) 2002-0037]; lipopolysaccharide (Sigma), MTT and nitric oxide determination kit (Biyuntian Biotechnology Co., Ltd.). Methods: The cortical astrocytes of neonatal rats were isolated and cultured in vitro according to the method of Kevin St. McNaught et al. After repeated subculturing and purification, the astrocytes were divided into 5 groups according to the dosage of lipopolysaccharide added: blank In the control group, lipopolysaccharide 5 mg / L group, lipopolysaccharide 10 mg / L group, lipopolysaccharide 20 mg / L group and lipopolysaccharide 40 mg / L group, the action time of lipopolysaccharide on astrocytes was 30 min and 60 min. The cell viability was measured by MTT assay at 30min and 60min respectively. The content of nitric oxide in 30min supernatant of lipopolysaccharide-treated cells was measured by Griess method. The effects of different doses of lipopolysaccharide on cells in vitro were observed by laser confocal microscopy. Astrocytes intracellular calcium, the role of reactive oxygen species level 60min when the relative fluorescence intensity percentage as a percentage of astrocytes within the calcium ion concentration and cell release of reactive oxygen species. MAIN OUTCOME MEASURES: Survival rate of astrocytes in each group; content of nitric oxide in astrocyte supernatant; changes of intracellular free calcium and reactive oxygen species in astrocytes under laser scanning confocal microscopy. Results: ① The lipopolysaccharide-induced astrocyte 60min, the cell viability of lipopolysaccharide 5mg / L, 10mg / L, 20mg / L group were increased, compared with the control group, the difference was significant (P <0.05 ), And the cell viability of lipopolysaccharide 10 mg / L group was higher than that of lipopolysaccharide 5 mg / L group and lipopolysaccharide 20 mg / L group. (2) The level of nitric oxide was increased 30min after treated with 40mg / L lipopolysaccharide, which was significantly different from the control group and the other three dose groups (P <0.05). ③ The relative fluorescence intensity of intracellular free calcium in LPS group 5mg / L, 10mg / L, 20mg / L group was 200 ~ 400 times of their corresponding basal state, and the fluorescence intensity of lipopolysaccharide 40mg / L group was the corresponding basic state L, 5 mg / L, 10 mg / L, and 20 mg / L of lipopolysaccharide were 200-300 times of those of basal state, but the absolute fluorescence intensity peak of lipopolysaccharide 40 mg / L group exceeded that of test Of the range, speculated that lipopolysaccharide 40mg / L group of reactive oxygen species level is at least 300 ~ 400 times the corresponding basal state. CONCLUSION: Lipopolysaccharide can increase the concentration of intracellular calcium and the release of ROS and NO, and promote cell proliferation after acting on astrocytes.