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背景:胆固醇可使体外培养的神经元自身突触数量增加,囊泡数量及递质释放效能亦明显增高。目的:建立大鼠脑海马组织中微量胆固醇的高效液相色谱检测方法,观察胆固醇在大鼠脑海马发育和成熟中的作用。设计:重复测量实验。材料:实验于2004-01/06在武汉大学人民医院神经科学研究室完成。选择健康雄性Wistar乳鼠(出生24h内)、幼鼠(28～30d,体质量40～60g)和成鼠(90～100d,体质量200～250g)各10只。方法:大鼠在麻醉状态下断头取海马称重,制备匀浆,离心,过滤后采用美国安捷伦公司反相色谱柱(ZORBAXXDBC184.6250mm5μm,Agilent),以乙腈-异丙醇(4∶1)为流动相;流速0.8mL/min;检测波长208nm。VARIANProstar325紫外检测器和美国VARIAN公司的LCWORKSTATIONV6.2色谱数据处理系统检测和处理色谱数据,获得胆固醇色谱图。样品中胆固醇含量(mg/g)=10×0.9965×样品中胆固醇的峰面积×标准胆固醇的含量(g/L)÷标准胆固醇的峰面积主要观察指标:①乳鼠、幼鼠和成鼠脑海马胆固醇含量与其色谱峰面积的关系。②乳鼠、幼鼠和成鼠脑海马胆固醇含量。结果:30只大鼠均进入结果分析。①胆固醇含量在0.1323～2.6472g/L时与其色谱峰面积呈良好的线性关系(r=0.99993)。②乳鼠、幼鼠和成鼠脑海马胆固醇含量:3组样品的胆固醇含量分别为(2.92±0.03),(11.20±3.41),(12.91±1.25)mg/g,差别均有极显著性意义(P<0.01)。结论:用高效液相色谱法检测大鼠脑海马组织中的微量胆固醇灵敏,特异性好,且分析时间短。在大鼠发育过程中,脑海马中胆固醇含量渐增,说明胆固醇对大鼠脑海马结构、功能的发育和成熟可能发挥了关键性的作用。
Background: Cholesterol can increase the number of synapses in vitro and the number of vesicles and the release efficiency of neurotransmitters. Objective: To establish a method for the determination of trace cholesterol in rat hippocampus by HPLC and to observe the effect of cholesterol on the development and maturation of hippocampus in rat brain. Design: Repeat measurement experiment. MATERIALS: The experiment was performed at the Department of Neuroscience, Wuhan University People’s Hospital from January to June 2004. Healthy male Wistar suckling mice (within 24 hours of birth), 10 young rats (28-30 days, 40-60 grams of body weight) and 10 rats (90-100 days, 200-250 grams of body weight) were selected. METHODS: The rats were weighed and decapitated in the anesthetizing state, homogenized and centrifuged. After being filtered, the samples were separated on a Agilent USA column (ZORBAXXDBC184.6250mm5μm, Agilent) with acetonitrile-isopropanol (4: 1) As mobile phase; flow rate 0.8mL / min; detection wavelength 208nm. VARIANProstar325 UV detector and VARIAN’s LCWORKSTATIONV6.2 chromatographic data processing system detects and processes chromatographic data to obtain a cholesterol chromatogram. Cholesterol content in the sample (mg / g) = 10 × 0.9965 × peak area of cholesterol in the sample × standard cholesterol (g / L) ÷ peak area of standard cholesterol Main Observations: Relationship between Horse Cholesterol Content and Its Peak Area. ② neonatal rats, young rats and rats brain hippocampus cholesterol content. Results: All 30 rats were involved in the result analysis. ① Cholesterol content showed a good linear relationship (r = 0.99993) with the chromatographic peak area at 0.1323 ~ 2.6472g / L. (2) Cholesterol content in hippocampus of suckling mice, young rats and adult rats: The cholesterol contents in the three groups were (2.92 ± 0.03), (11.20 ± 3.41) and (12.91 ± 1.25) mg / g, respectively, with significant differences (P <0.01). Conclusion: The detection of trace cholesterol in rat brain hippocampus by high performance liquid chromatography is sensitive and specific, and the analysis time is short. During the development of rats, the content of cholesterol in hippocampus gradually increased, indicating that cholesterol may play a key role in the development and maturation of brain hippocampus.