朊蛋白106—126肽段在Prion病细胞模型中的作用机制研究

Cytotoxic effects of differentiated PC12 cell infected by prion protein 106 - 126 peptide

目的研究朊蛋白106—126肽段在细胞水平上的毒性作用机制。方法PCI2细胞常规培养后加入神经生长因子（NGF）诱导成神经元样细胞，再加入朊蛋白106—126肽段，检测细胞存活率，观察细胞生长和形态变化，检测细胞氧化应激现象和能量代谢的改变，检测细胞凋亡现象及其相关机制。结果细胞接触肽段后存活率由（98．1±1．9）％下降到（69．2±4．7）％，流式细胞仪检测细胞出现亚二倍体峰。氧化应激现象于接触肽段12h后出现并且持续存在，细胞内钙离子浓度从（185．74±12．93）nmol／L增加到（493．00±58．71）nmol／L，线粒体膜电位下降至65％，Ca^2＋ATP酶活性从54．92±4．05降低至34．92±4．86，细胞能量代谢水平下降，最终出现细胞凋亡，Bcl-2／Bax系统稳态变化参与了诱导凋亡的过程。结论利用106—126肽段感染分化的PCI2细胞可能是在细胞水平上研究朊蛋白毒性作用的理想模型。PrP106—126的毒性作用是通过持续的氧化应激、干扰细胞的能量代谢、钙超载、最终诱导宿主细胞的凋亡来实现的，并且氧化应激可能处于始动与核心的地位。

Objective To investigate the cytotoxic effects of differentiated PC12 cells after infected by prion protein 106 - 126 peptide. Methods The PC12 cells were infected by prion protein 106 - 126 peptide after differentiated by nerve growth factor （NGF）. Cell viability and the morphological changes were observed. The energy metabolize and apoptosis was detected. Results After infected by this peptide, cell viability decreased from （98. 1 ± 1.9） % to （69. 2 ± 4. 7 ） %, and apoptosis peak was observed by flow cytometry. About the process of the cytotoxic effects, after the cells affected by PrP106 - 126, oxidative stress presented and existed continually, and then the intracellular free calcium concentrate increased from （ 185.74±12. 93 ） nmol/L to （ 493.00±58.71 ） nmol/L subsequently, the activity of Ca^2 ＋ ATPase decreased from 54. 92 ± 4. 05 to 34.92 ± 4. 86, the mitochondrial membrane potential decreased to 65%, and also the energy metabolize disorder, the cells presented apoptosis in the end. The changed Bcl -2/Bax system involved in the apoptosis. Conclusions Prion protein 106 - 126 peptide can induce apoptosis in differentiated PC12 cells and presented cellular toxicity definitely. It might be a perfect model to study the cellular toxicity of prion protein. Continual oxidative stress could cause the intracellular free calcium concentrate and disturb the energy metabolize, and the apoptosis might be the end-result. The oxidative stress of might play a startup and important role.