酒精诱导PC12细胞自噬及其与P62作用的关系-护理学专业论文.docxVIP

I I 摘 要 目的 采用不同浓度酒精作用于大鼠嗜铬细胞瘤细胞(PC12 细胞)，观察细胞自噬发生及其 与 P62 变化的关系。 方法 用四甲偶氮唑盐比色法(MTT)观察酒精对 PC12 细胞生存率的影响；间接免疫荧光 法检测细胞自噬标志性蛋白 LC3 和 P62 的变化；高内涵活细胞成像系统检测细胞 LC3 荧光强度；透射电镜检测细胞自噬的超微结构；Western blotting 方法检测 P62 蛋白量的 表达。 结果 100 mmol/L、200 mmol/L、400 mmol/L 和 800 mmol/L 浓度酒精处理 PC12 细胞 24 h， 与 0 mmol/L 酒精组比较，PC12 细胞生存率分别为 91.97% (P＜0.05)、72.63% (P＜0.01)、 58.23% (P＜0.01)、50.82% (P＜0.01)，酒精对 PC12 细胞的增殖有显著的抑制作用，呈 浓度依赖性。间接免疫荧光检测到酒精致 PC12 细胞自噬标志性蛋白 LC3 在细胞核周围 密度增高，并与 P62 形成点状聚集共定位；高内涵活细胞成像系统检测到酒精浓度为 200 mmol/L 作用 2 h 时，PC12 细胞 LC3 自噬荧光强度最高；透射电镜也观察到酒精作 用的 PC12 细胞质中自噬体和自噬溶酶体。Western blotting 结果显示，不同浓度酒精处 理 PC12 细胞 2 h，P62 蛋白表达量显著增加 (P＜0.01)；用 200 mmol/L 浓度酒精处理 PC12 细胞，P62 蛋白表达在 2 h 达到最高值。 结论 酒精诱导 PC12 细胞的发生自噬现象，P62 蛋白参与此自噬调控过程。 关键词：酒精，自噬，P62，LC3，PC12 细胞 III III IV IV ABSTRACT Objective The study utilized pheochromocytoma PC12 cells to examine the effect of ethanol on autophagy and the role of P62 in ethanol-induced autophagy. Methods The inhibition effect of ethanol on proliferation of PC12 cells was examined by MTT assay. Indirect immunofluorescence was used to detect common location of LC3, a biomarker of autophagy, and P62. The LC3 fluorescence intensity in cellular cytosol was measured using a high content screening imaging system. The ultrastructural morphology of autophagic vacuoles and autophagy-lysosome were observed under the transmission electron microscope. Protein expression of P62 was detected with Western blotting analysis. Results Ethanol at the concentration of 100 mmol/L, 200 mmol/L, 400 mmol/L and 800 mmol/L decreased cell proliferation of PC12 cells to 91.97% (P＜0.05), 72.63% (P＜0.01), 58.23% (P＜0.01), 50.82% (P＜ 0.01) respectively and these groups were compared to the control in serum-free medium. The study indicated a dose-dependent inhibitory effect of ethanol on cell proliferation．Indirect immunofluorescence staining and the high content screening imaging system showed that ethanol increased LC3 fluorescence intensity and the co-localization of LC3 with P62 in