小鼠脑片Formazan定量测量方法评价缺血性损伤及神经保护作用  

A quantitative measurement of formazan for evaluating ischemic injury and neuroprotection in mouse brain slices

作　　者：葛求富[1] 魏尔清[1] 彭国平[1] 于丽芬 余国良[1] 刘建仁[1] 

机构地区：[1]浙江大学医学院药理学教研室,杭州310031 [2]杭州民生药业股份有限公司,杭州310011

出　　处：《中国药理学通报》2003年第10期1102-1106,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助 (No 3 0 2 714 98);浙江省自然科学基金资助(No 3 990 90 )

摘　　要：目的　建立一种定量测量 2 ,3,5 三苯基四氮唑(TTC)红色还原产物formazan的方法 ,用于评估离体脑片缺血性损伤以及依达拉奉和ONO 10 78的神经保护作用。方法　以TTC为底物在小鼠脑片生物合成formazan标准品 ,并进行分离、纯化和鉴定。小鼠脑片以缺氧缺糖 (OGD)方法诱导损伤 ,用分光光度法在 4 90nm处测量皮质和纹状体formazan合成量。依达拉奉和ONO 10 78加入培养液 ,观察其神经保护作用。结果　合成并纯化了formazan标准品 ,其纯度为 0 993,线性测量范围在 0 0 5～ 1g·L-1。OGD减少formazan合成 ,与处理时间有依赖性。依达拉奉 (0 0 1～ 1μmol·L-1)抑制OGD诱导的formazan合成减少 ,而ONO 10 78无作用。结论　定量测量formazan是一种评价离体脑片损伤及药物神经保护作用的有用方法。AIM To establish a quantitative method for measuring formazan, a product of 2,3,5 triphenyltetrazolium chloride (TTC), and use it to evaluate in vitro ischemic injury and neuroprotective effects of edaravone and ONO 1078 in mouse brain slices. METHODS Standard formazan was biosynthesized in mouse brain slices using TTC as the substrate and then isolated, purified, and identified. After mouse brain slices were treated by oxygen/glucose deprivation (OGD) to induce injury, the formazan product in the cortex and striatum was measured and calculated based on the absorbance of the formazan standard solution. Edaravone and ONO 1078 were added into the incubation media to observe their neuroprotective effects. RESULTS Standard formazan was biosynthesized and purified with a purity of 0 993 and a linear range of 0 05～1 0 g·L -1 . OGD decreased formazan product in a duration dependent manner. Edaravone (0 01～1 μmol·L -1 ) recovered the formazan product decreased by OGD, but ONO 1078 had no effect. CONCLUSION The quantitative measurement of formazan is a useful method for evaluating brain injury and neuroprotective effects of drugs in brain slices in vitro.

关 键 词：氯化2 3 5-三苯基四氮唑 FORMAZAN 缺氧缺糖 小鼠脑片 脑缺血 依达拉奉 MCI-186 3-甲基-1-苯基-2-吡唑啉-5-酮基 ONO-1078{pranlukast 4-氧-8-[对-(4-苯丁氧基)苯甲酰氨基]-2-(5-四氮基)-4H-1-苯并吡喃半水化合物) 

分 类 号：R-332[医药卫生] R322.81