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用不同浓度的亚硒酸钠加入原代培养两天的新生小鼠大脑皮质神经细胞 ,在不同的作用时间用分子生物学和流式细胞仪技术观察了亚硒酸钠诱导离体神经元凋亡的时间过程和量 效反应关系 ;同时 ,进一步观察了亚硒酸钠引发凋亡时一些相关基因表达的改变。结果显示 :①提取对照组和不同剂量亚硒酸钠处理 2、4、2 4和 48h组的DNA片段 (每个样品取 3× 10 6细胞 )进行琼脂糖凝胶电泳 :除对照组外 ,在亚硒酸钠处理的几个时间点都出现了神经元凋亡的特征性改变—DNA梯型 ,有剂量 效应和效应 时间依赖性 ,而以 0 .5 μmol/L亚硒酸钠处理后 2 4h表现得最明显 ;这些改变在同时加入核酸内切酶抑制剂ATA时消失或有不同程度的减弱 ;②用流式细胞仪对每个样品分析 10 0 0 0细胞得到的DNA含量直方图表明 ,亚二倍体峰(即以“AP”标出的凋亡峰 )的出现与亚硒酸钠呈明显的剂量和时间依赖性 ,其变化趋势与琼脂糖凝胶电泳的结果相吻合 ;③用RT PCR检测技术 (提取总RNA后 ,用反转录系统将其制备成cDNA ,再用特异性引物经PCR扩增要检测的cDNA后作半定量分析 )显示 ,在亚硒酸钠处理后的不同时间 ,与细胞凋亡密切相关的bcl 2、bax、p5 3、c fos、和AChE几种基因转录的mRNA量出现不同方向的改变 :bcl 2mRNA的量在亚硒酸钠处理后 1h明
With different concentrations of sodium selenite added two days of primary cultured neonatal mouse cerebral cortical neurons at different time points by molecular biology and flow cytometry technology observed sodium selenite-induced in vitro neuronal apoptosis The course of death and the dose-response relationship. At the same time, we further observed the change of some related gene expression induced by sodium selenite. The results showed that: (1) The DNA fragments (3 × 10 6 cells per sample) of the control group and the groups treated with different doses of sodium selenite for 2, 4, 24, and 48h were extracted and subjected to agarose gel electrophoresis. Except for the control group, At several time points of sodium selenite treatment, a characteristic change in neuronal apoptosis was observed-DNA ladder with dose-dependent and time-dependent effects, whereas treatment with 0.5 μmol / L sodium selenite 2 4h showed the most obvious; these changes in the simultaneous addition of endonuclease inhibitor ATA disappeared or with varying degrees of attenuation; ② flow cytometry for each sample analysis of 1000 cells obtained DNA content histogram The results showed that the appearance of sub-diploid peak (that is, the peak of apoptosis marked by “AP”) was in a dose-and time-dependent manner with sodium selenite, and the trend was consistent with the result of agarose gel electrophoresis. ③ RT PCR detection technology (extracted total RNA, the reverse transcription system to prepare it into cDNA, and then use specific primers to be detected by PCR amplification of cDNA semi-quantitative analysis) showed that in the sodium selenite treatment After different time, closely related to apoptosis of bcl 2, bax, p5 3, c fos , And AChE mRNA transcripts in different directions appeared in different directions: the amount of bcl 2 mRNA after 1 h sodium selenite treatment