摘要
目的 :通过改进和完善脑片孵育装置、脑片缺血性损伤的定量指标 ,建立一种简单、准确评价小鼠脑片缺血性损伤及药物神经保护作用的新方法。方法 :设计和制作脑片孵育装置 ,并验证其适用性。以 TTC为底物在小鼠脑片生物合成 formazan标准品 ,并作分离、纯化和鉴定。以缺氧缺糖 (OGD)方法诱导小鼠脑片损伤 ,用分光光度法在 4 90 nm处测量皮质和纹状体 formazan量 ,并观察依达拉奉和 ONO- 10 78的保护作用。结果 :脑片孵育装置具有良好的平行性和可操作性。获得了纯度为 99.3%的 formazan对照品 ,在 0 .0 5～ 1mg/ml间与 4 90 nm的吸收度有良好的线性关系 (r=0 .9997)。随 OGD时间延长 ,脑片皮质和纹状体 formazan量逐渐减少 ;依达拉奉对此有显著保护作用 ,而 ONO- 10 78无效。结论 :脑片孵育新装置及 formazan定量方法效率高、定量准确、简单易行 ,可用于缺血性脑损伤保护药物的筛选。
Objective: To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices. Methods: An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices,and formazan was isolated,purified and identified.Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD),the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects. Results: The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently.Standard formazan was biosynthesized and purified with a purity of 99.3%,and showed a linear range of 0.05～1 mg/ml in absorbance at 490 nm (r=0.9997).OGD decreased formazan production in the cortex and striatum in a duration-dependent manner.Edaravone (0.01 to 1 μmol/L) recovered OGD-induced decrease of formazan production,but ONO-1078 showed no effect. Conclusion: The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2003年第6期486-491,共6页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金 (30 2 714 98)
浙江省自然科学基金 (3990 90 )资助项目
