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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。
Background: High concentrations of nitric oxide cause neuronal apoptosis. Therefore, inhibition of nitric oxide-induced apoptosis can achieve the purpose of prevention and treatment of Alzheimer's disease. Objective: To observe whether acidic peptide can inhibit nitric oxide-induced neuronal apoptosis. Design: Controlled observation of cellular and molecular experiments. MATERIALS: The experiment was performed at the Laboratory of Bioactive Peptide Research, Zhengzhou University from May 2003 to May 2005 and the cell culture room of the Department of Biochemistry and Molecular Biology, School of Basic Medicine, Zhengzhou University. SD neonatal rats within 24h, provided by the Henan Animal Center (410117). Methods: Primary cultured hippocampal neurons of neonatal SD rats were cultured. The nerve cells cultured on the 11th day were pretreated with different doses of acidic peptide for 6h, and then added with a final concentration of 50μmmol / L of nitrosylated sodium cyanide. The cells were cultured for another 24h, and the cells were harvested for experiments. Each experiment was divided into the following five groups: normal control group, nitrosylated sodium cyanide treatment group, nitrosylated sodium cyanide + 0.0375mg / mL acidic peptide group, nitrosylated sodium cyanide + 0.075mg / mL acidic peptide group, nitroso-sodium ferrocyanide + 0.15 mg / mL acidic peptide group. Cell viability was measured by thiazolyl blue staining, neurofilament protein was stained with immunohistochemical method, and then apoptotic morphology was observed by acridine orange fluorescence staining. DNA ladder of apoptotic cells was analyzed by agarose gel electrophoresis. The expression levels of Bcl-2 protein and Bax protein were analyzed by Western blot and absorbance scanning. MAIN OUTCOME MEASURES: ① Experimental results of cell viability assay by thiazolyl blue colorimetry. ② karyotype observation of apoptosis. ③ DNA electrophoresis analysis of apoptosis. ④ Western Blot analysis of Bcl-2 and Bax proteins. Results: ① The survival rate of neurons was 58.9% in the group of nitrosylated sodium cyanide, 70.0% in the group of nitrosylated sodium cyanide + 0.037 mg / mL of acid peptide, and the percentage of nitrosylated sodium ferricyanide +0.075 72.8% in the group of mg / mL acidic peptide and 75.3% of the group treated with 0.15% nitrosyl sodium cyanide. ② karyotype observation of apoptosis: nitrosylated sodium cyanide treatment group showed obvious characteristics of apoptosis. The nuclei of hippocampal neurons co-treated with different concentrations of acidic peptides + sodium nitrite were close to those of normal control. (3) The results of DNA electrophoresis analysis of apoptosis showed that the DNA of neurons in the group treated with nitrosylated sodium cyanide only showed characteristic apoptotic DNA ladder on agarose gel electrophoresis. Western Blot and absorbance scanning analysis of Bcl-2 and Bax protein showed that the expression of Bcl-2 protein and Bax protein in nitrosylated sodium cyanide treatment group decreased. However, the Bcl-2 protein levels of acidic peptide plus sodium nitrosofenac sodium co-treated with different concentrations gradually increased with the concentration of acidic peptide and the expression of Bax protein decreased gradually. Conclusion: Acidic peptide can inhibit neuronal apoptosis, increase the expression of Bcl-2 protein and inhibit the expression of Bax protein in neurons.