【摘要】 背景:高浓度的一氧化氮能引起神经细胞的凋亡。因此抑制一氧化氮引起的细胞凋亡就能达到预防和治疗老年痴呆病的目的。目的:观察酸性肽能否抑制一氧化氮引起的神经元凋亡。设计:对照观察细胞和分子实验。材料:实验于2003-05/2005-05在郑州大学生物活性肽研究所第二实验室和郑州大学基础医学院生物化学与分子生物学教研室细胞培养室完成。新生的24h内SD雄性大鼠,由河南省动物中心提供(410117)。方法:对新生的SD大鼠海马神经元进行原代培养。取培养至第11天的神经细胞,用不同剂量的酸性肽预处理6h,再加入终浓度为50μmmol/L的亚硝基铁氰化钠,继续培养24h,收集细胞进行实验。每次实验均分为以下5组:正常对照组、亚硝基铁氰化钠处理组、亚硝基铁氰化钠+0.0375mg/mL酸性肽组、亚硝基铁氰化钠+0.075mg/mL酸性肽组,亚硝基铁氰化钠+0.15mg/mL酸性肽组。用噻唑蓝法测定细胞的存活率,用免疫组织化学法对神经丝蛋白进行染色,再用吖啶橙荧光染色法显示细胞凋亡的形态。用琼脂糖凝胶电泳法分析凋亡细胞的DNA梯带。用WesternBlot和吸光度扫描分析Bcl-2蛋白和Bax蛋白的表达水平。主要观察指标:①噻唑蓝法比色法检测细胞存活率的实验结果。②细胞凋亡的核型观察结果。③细胞凋亡的DNA电泳分析。④Bcl-2和Bax蛋白的WesternBlot分析结果。结果:①亚硝基铁氰化钠处理组的神经元存活率为58.9%,亚硝基铁氰化钠+0.037mg/mL酸性肽处理组为70.0%,亚硝基铁氰化钠+0.075mg/mL酸性肽处理组为72.8%,亚硝基铁氰化钠+0.15mg/mL酸性肽处理组为75.3%。②细胞凋亡的核型观察结果:亚硝基铁氰化钠处理组呈现细胞凋亡的明显特征。不同浓度的酸性肽+亚硝基铁氰化钠共同处理组海马神经元的细胞核接近正常对照组的细胞核形态。③细胞凋亡的DNA电泳分析结果表明,仅亚硝基铁氰化钠处理组的神经元DNA在琼脂糖凝胶电泳上显示出清晰的细胞凋亡的特征性DNA梯带。④Bcl-2和Bax蛋白的Western Blot和吸光度扫描分析结果显示,亚硝基铁氰化钠处理组Bcl-2蛋白表达水平下降,Bax蛋白表达水平升高。而不同浓度的酸性肽+亚硝基铁氰化钠共同处理组的Bcl-2蛋白水平随酸性肽浓度逐渐增加,Bax蛋白的表达水平逐渐下降。结论:酸性肽能够抑制神经元的凋亡,增加神经元Bcl-2蛋白的表达水平,抑制神经元Bax蛋白的表达水平。更多还原

【Abstract】 BACKGROUND:Excessive nitric oxide (NO) release can cause the occurrence and development of brain injury and senile dementia due to the apoptosis induction role of NO at high concentration to nerve cells. Therefore one strategy to prevent and treat senile dementia is inhibiting the apoptosis induced by NO. OBJECTIVE:To observe whether acidic peptide will inhibit the neuron apoptosis caused by NO. DESIGN:An cell and molecule observation experiment by comparisons. SETTING:Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University and the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University. MATERIALS:The experiment was performed between May 2003 and May 2005,in the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University and the cell culture room of Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University. The newborn SD male rats within 24 hours after birth were provided by the Animal Center of Henan Province (410117). METHODS:On day 11 of primary cultures,hippocampus neurons of the newborn SD rats were pretreated with different dosages of acidic peptide for six hours. Sodium nitroprusside (SNP) of 50 μmol/L final concentration was added to the cells which were incubated for another 24 hours. Cells were collected and adopted in this experiment of five different groups,namely normal control group,group treated with SNP,group of SNP plus 0.037 5 mg/mL acidic peptide,group of SNP plus 0.075 mg/mL acidic peptide,group of SNP plus 0.15 mg/mL acidic peptide. The cell’s survival rate was measure by methyl thiazolyl (MTT) method; The neurofilament protein was stained with the method of immunohistochemistry. The shape of apoptosis was display with acridine orange fluorescent stain. Then DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Western Blot and absorbance scan were used to determine the expression level of Bcl-2 protein and Bax protein. MAIN OUTCOME MEASURES:①Experimental result of cell survival rate with MTT method;②Observation results of nuclear type of apoptosis;③DNA electrophoresis analysis of apoptosis;④Western Blot analysis results of Bcl-2 protein and Bax protein. RESULTS:①Neuron survival rate was 58.9% for group treated with SNP,70.0% for group of SNP plus 0.037 mg/mL acidic peptide,72.8% for group of SNP plus 0.075 mg/mL acidic peptide,and 75.3% for group of SNP plus 0.15 mg/mL acidic peptide.②Observation results of nuclear type of apoptosis:Significant characteristics of apoptosis were seen in group treated with SNP. The nucleus of hippocampus neuron treated with different concentrations of acidic peptide plus SNP was similar to that of normal control group in morphology.③The results of DNA electrophoresis analysis of apoptosis:Only the neuron DNA of group treated with SNP showed clear characteristic DNA ladder zone of apoptosis on agarose gel electrophoresis.④Analysis results of Bcl-2 protein and Bax protein with Western Blot and absorbance scan:The expression level of Bcl-2 protein in SNP treated group was decreased while that of Bcl-2 protein was increased. Bcl-2 protein levels in acidic peptide plus SNP group were increased and Bax protein levels were decreased gradually with the increasing concentrations of acidic peptide compared with SNP treated group. CONCLUSION:Acidic peptide can inhibit neuron apoptosis,increase expression level of neuron Bcl-2 protein and decrease expression level of neuron Bax protein.更多还原