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在过去工作证实人工合成的β淀粉样蛋白片段31~35(Aβ31~35)诱发体外培养的新生小鼠大脑皮质神经元凋亡的基础上,本研究探讨了这一凋亡的详细时程和一些相关基因表达的变化。结果显示:(1)用荧光细胞核染料PI染色,观察到Aβ31~35 处理后1 h 开始出现神经元核染色质凝聚,2 h 可看到核沟出现和大小不等的核片块,在3,4 和24 h 出现越来越多的核片块,但不同细胞核的变化不同步,即有些细胞出现核损害的同时,也有核正常的细胞存在;(2)用琼脂糖凝胶电泳分析各时间点提取的DNA,梯型现象最早出现在Aβ31~35 处理后2 h,随着时间延长越来越明显;(3)用流式细胞仪检测DNA 含量直方图显示,亚二倍体细胞最早出现在Aβ31~35 处理后2 h,其数量随时间延长逐渐增多;(4)用免疫荧光流式细胞仪检测某些基因编码的蛋白产物的表达量表明,bcl2 蛋白表达在Aβ31~35 处理后1 h 出现明显减少,以后缓慢降低,至24 h达最低点。与此相反,bax 蛋白表达在1~4 h 期间呈现稳定上调,而p53 和cfos 两种蛋白表达也显示上调,其p53 蛋白在2 h 达最高峰,cfos 蛋白则在2 h 开始上调;(5)用RTPCR
In the past work confirmed that synthetic β-amyloid protein fragments 31 ~ 35 (Aβ31 ~ 35) induced in vitro cultured neonatal mouse cerebral cortex neurons on the basis of apoptosis in this study to explore the detailed timing of this apoptosis And some related gene expression changes. The results showed that: (1) PI staining of fluorescent nuclear dye PI showed that nuclear chromatin condensation began to occur at 1 h after Aβ31-35 treatment, and nuclei blocks of different sizes appeared in 2 h. , 4 and 24 h appeared more and more nuclear pieces, but different nuclei changes are not synchronized, that is, some of the cells in nuclear damage, there are nuclear normal cells; (2) by agarose gel electrophoresis analysis DNA and trapezoidal DNA extracted at the time point first appeared 2 hours after treatment with Aβ31-35, which became more and more obvious with time. (3) Histograms of DNA by flow cytometry showed that the earliest diploid cells (4) The expression level of some protein encoded by some genes detected by immunofluorescence flow cytometry indicated that the expression of bcl2 protein was in the range of Aβ31 ~ 35 At 1 h after treatment, there was a significant decrease, then slowly decreased to the lowest point at 24 h. In contrast, the expression of bax protein showed a steady increase during 1 ~ 4 h, and p53 and c fos expression of two proteins also showed up, the peak of its p53 protein at 2 h, c fos protein began at 2 h Up; (5) RT PCR