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目的系统地研究PrP106-126神经毒性多肽对原代神经元造成的损伤。方法使用出生24 h内的SD乳鼠分离培养原代神经元,经过PrP106-126处理后,利用免疫印迹技术检测凋亡相关蛋白的变化;利用透射电镜观察功毒后原代神经元亚细胞结构的变化情况;利用流式细胞仪检测细胞活性;利用免疫荧光技术观察细胞凋亡情况。结果随着PrP106-126刺激原代神经元的时间延长,抑制凋亡蛋白的表达量不断下降,促凋亡蛋白的表达量不断升高;透射电镜显示,经过多肽刺激的原代神经元的细胞器结构有不同程度的损伤,细胞胞浆内出现大小不等的单层或多层空泡结构;TUNEL细胞凋亡检测和Annexin V-FITC细胞活力检测试剂盒的检测结果都表明,经多肽刺激后,原代神经元的细胞活力显著下降,并有大量神经元发生凋亡。结论 PrP106-126神经毒性多肽激活促凋亡信号通路,造成细胞内的平稳紊乱和超微结构损伤,诱导原代神经元发生凋亡,为朊病毒疾病研究模型的建立提供全面可靠的证据。
Objective To systematically study the damage of primary neurons caused by PrP106-126 neurotoxic peptides. Methods Primary neurons were isolated from SD suckling mice within 24 hours of birth. After PrP106-126 treatment, the changes of apoptosis related proteins were detected by Western blotting. Transmission electron microscopy was used to observe the primary neuronal subcellular structures The changes of cell viability were detected by flow cytometry. The apoptosis of cells was observed by immunofluorescence staining. Results With PrP106-126 stimulation of primary neurons prolonged, the expression of anti-apoptotic protein decreased and the expression of pro-apoptotic protein increased. Transmission electron microscopy revealed that the organelles of primary neurons stimulated by peptide The structure has varying degrees of damage, the cytoplasm appeared within the cytoplasm of single or multiple layers of vacuolar structure; TUNEL apoptosis and Annexin V-FITC cell viability test kit test results show that after stimulation by the polypeptide , Primary neurons significantly decreased cell viability, and a large number of neurons apoptosis occurred. CONCLUSIONS: PrP106-126 neurotoxicity peptide activates the pro-apoptotic signaling pathway, resulting in stable intracellular disturbance and ultrastructure damage, inducing primary neuron apoptosis and providing comprehensive and reliable evidence for the establishment of a prion disease research model.