CAJ | 학술논문

目的利用建立好的转染A53T突变型α-突触核蛋白的大鼠嗜铬细胞瘤(PC12)细胞株,探讨自噬和泛素-蛋白酶体通路在细胞凋亡途径中的具体作用.方法选择特异性蛋白酶体抑制剂和大自噬抑制剂及诱导剂作用于稳定转染的A53T细胞株,四甲基偶氮唑盐法和流式细胞仪检测细胞活力和各组细胞凋亡率,电镜观察超微结构,并测定细胞培养液中NO的活力和热休克蛋白70(Hsp70)及Caspase-3蛋白表达.结果蛋白酶体抑制剂环氧霉素(100 nmol/L)和(或)大自噬抑制剂3-MA(10 mmol/L)处理A53T细胞24 h后环氧霉素组、3-MA组和环氧霉素+3-MA组细胞活力(A值分别为0.23±0.01、0.19±0.01、0.17±0.01)较对照A53T细胞组(A值为0.32±0.06)明显下降(P<0.05);而大自噬诱导剂雷帕霉素(0.2 μg/ml)处理后细胞存活率(A值为0.44±0.08)显著高于其余用药组.与对照组(1.55%±1.15%)相比,3-MA、环氧霉素、环氧霉素+3-MA组作用24 h后A53T细胞的凋亡百分率(分别为4.74%±0.91%、4.59%±1.18%、5.40%±1.75%)显著增高(P<0.05),而大自噬诱导剂下调蛋白酶体抑制剂导致的凋亡;蛋白酶体抑制剂组NO和Hsp70蛋白含量也明显高于对照组.结论大自噬和蛋白酶体途径障碍促进了凋亡发生,抑制或诱导自噬对Hsp70和NO的影响不如蛋白酶体途径对其影响大.
목적 이용건립호적전염A53T돌변형α-돌촉핵단백적대서기락세포류(PC12)세포주,탐토자서화범소-단백매체통로재세포조망도경중적구체작용.방법 선택특이성단백매체억제제화대자서억제제급유도제작용우은정전염적A53T세포주,사갑기우담서염법화류식세포의검측세포활력화각조세포조망솔,전경관찰초미결구,병측정세포배양액중NO적활력화열휴극단백70(Hsp70)급Caspase-3단백표체.결과 단백매체억제제배양매소(100 nmol/L)화(혹)대자서억제제3-MA(10 mmol/L)처리A53T세포24 h후배양매소조、3-MA조화배양매소+3-MA조세포활력(A치분별위0.23±0.01、0.19±0.01、0.17±0.01)교대조A53T세포조(A치위0.32±0.06)명현하강(P<0.05);이대자서유도제뢰파매소(0.2 μg/ml)처리후세포존활솔(A치위0.44±0.08)현저고우기여용약조.여대조조(1.55%±1.15%)상비,3-MA、배양매소、배양매소+3-MA조작용24 h후A53T세포적조망백분솔(분별위4.74%±0.91%、4.59%±1.18%、5.40%±1.75%)현저증고(P<0.05),이대자서유도제하조단백매체억제제도치적조망;단백매체억제제조NO화Hsp70단백함량야명현고우대조조.결론 대자서화단백매체도경장애촉진료조망발생,억제혹유도자서대Hsp70화NO적영향불여단백매체도경대기영향대.
Objective To explore the specific role of autophagy and ubiquitin-proteasome pathway in apoptosis, specific protease inhibitor and (or) macroautophagy inhibitors.Methods The stimulators were selected to work on the pheochromocytoma (PC12) cell lines transfected with human mutant α-synuclein (A53T).Cell activity and apeptosis rate were detected by MTT law and flow cytometry.NO energy, heat shock protein 70 (Hsp70) and Caspase-3 expression were determined in cell culture.Results A53T cell survival rate significantly decreased 24 hours after handling with the protease inhibitor (100 nmol/L) and (or) autophagy inhibitors 3-MA (10 mmol/L, A =0.23±0.01,0.19±0.01 and 0.17±0.01 respectively; P <0.05) compared with the control group (A =0.32±0.06).Cell survival rate was significantly higher than the other drug group after 24 hours handling with autophagy stimulators (A =0.44±0.08).Compared with the control group or autophagy stimulator of rapamycin (0.2 μg/ml) group (1.55%±1.15%), A53T cells apeptosis percentage rate was significantly higher after treated with proteasome inhibitor and macroautophagy inhibitors 24 hours (4.74%±0.91%, 4.59%±1.18% and 5.40%±1.75%respectively, P <0.05); and a slight decrease with stimulators.Protein Hsp70 and NO were significantly higher in proteasome inhibitor groups than the control group.But in antophagy inhibitor and stimulator group, NO and Hsp70 protein was similar to the control group.Conclusion The inhibition of macroautophagy and proteasome can promote apoptosis.Inhibiting or stimulating autophagy has less impact on Hsp70 and NO than proteasome pathway.