CAJ | 학술논문

目的探讨沉默信息调控因子1(SIRTl)在脂多糖(LPS)诱导的PC12细胞凋亡过程中的作用. 方法 PC12细胞按不同培养方法分为6组:对照组、250 μg/mL组、500 μg/mL组、750μg/mL组、1000 μg/mL组、1250 μg/mL组；对照组常规培养,后5组添加相应浓度的LPS培养.24 h后MTT法测量细胞存活率,同时确定LPS诱导PC12细胞凋亡的适合浓度.再按选定的浓度培养细胞,并根据培养时间的不同分为6组:对照组、1/2 h组、2h组、18h组、24 h组、48 h组,Hoechst染色及流式细胞仪测量细胞凋亡率.Western blotting检测各组细胞中SIRT1的表达情况. 结果 Hoechst染色结果提示PCl2细胞经LPS培养1/2h后出现凋亡,表现为核固缩、核碎裂;18h开始凋亡小体增多,24h达到高峰,48 h后又有所下降.流式细胞仪检测结果显示各实验组细胞凋亡率与对照组相比差异均有统计学意义(P＜0.05),凋亡率变化趋势与Hoechst染色法观察到的结果一致.Western blotting结果显示对照组SIRTl表达量为1.84±0.04; 1/2 h时SIRT1蛋白表达减低至1.17±0.09;24 h时表达降至最低,为0.62±0.03;48 h组表达量有所回升,达到0.77±0.02;实验组各时间点组与对照组相比差异均有统计学意义(P＜0.05). 结论 LPS可以诱导PC12细胞凋亡,SIRT1在此过程中的表达受到抑制；推测SIRT1在LPS诱导PC12细胞凋亡的过程中起到一定的保护作用.
목적 탐토침묵신식조공인자1(SIRTl)재지다당(LPS)유도적PC12세포조망과정중적작용. 방법 PC12세포안불동배양방법분위6조:대조조、250 μg/mL조、500 μg/mL조、750μg/mL조、1000 μg/mL조、1250 μg/mL조；대조조상규배양,후5조첨가상응농도적LPS배양.24 h후MTT법측량세포존활솔,동시학정LPS유도PC12세포조망적괄합농도.재안선정적농도배양세포,병근거배양시간적불동분위6조:대조조、1/2 h조、2h조、18h조、24 h조、48 h조,Hoechst염색급류식세포의측량세포조망솔.Western blotting검측각조세포중SIRT1적표체정황. 결과 Hoechst염색결과제시PCl2세포경LPS배양1/2h후출현조망,표현위핵고축、핵쇄렬;18h개시조망소체증다,24h체도고봉,48 h후우유소하강.류식세포의검측결과현시각실험조세포조망솔여대조조상비차이균유통계학의의(P＜0.05),조망솔변화추세여Hoechst염색법관찰도적결과 일치.Western blotting결과현시대조조SIRTl표체량위1.84±0.04; 1/2 h시SIRT1단백표체감저지1.17±0.09;24 h시표체강지최저,위0.62±0.03;48 h조표체량유소회승,체도0.77±0.02;실험조각시간점조여대조조상비차이균유통계학의의(P＜0.05). 결론 LPS가이유도PC12세포조망,SIRT1재차과정중적표체수도억제；추측SIRT1재LPS유도PC12세포조망적과정중기도일정적보호작용.
Objective To study the role of SIRT 1 in apoptosis of PC 12 neuronal cells induced by lipopolysaccharide (LPS). Methods PC12 cells were cultured with different concentrations of LS (50 μg/mL,500 μg/mL,750 μg/mL,1000 μg/mL and 1250 μg/mL),and some other PC12 cells were routinely cultured as controls. MTT assay was employed to identify the cell survival 24 h after the inducement,and accordingly,the suitable LPS concentration for subsequent experiments was determined based on MTT results. And then, cell apoptosis in the experimental groups under the suitable LPS concentration at different times (1/2,2,18,24,and 48 h) and control group was noted by flow cytometry and Hoechst 33258 staining; Western blotting was used to detect the SIRT1 level in PC12 cells. Results Hoechst 33258 staining indicated that a few apoptotic bodies were noted 1/2 h after inducement,expressing as karyopyknosis and karyorrhexis; apoptotic bodies began to increase 18 h after inducement,reaching their peak level 24 h after inducement; and a decreased trend was observed 48 h after inducement. Flow cytometry indicated that significantly higher apoptosis rate at each time point was noted as compared with that in the control group (P＜0.05); and Hoechst 33258 staining showed the same result. Western blotting revealed that the SIRT1 expression was (1.84±0.04) in the control group,decreasing to (1.17±0.09) 1/2 h after the inducement,and reaching the lowest level (0.62±0.03) 24 h after the inducement; and then, the expression was increased to (0.77±0.02) 48 h after the inducement;significant difference on the expression at each time point was noted as compared with that in the control group (P＜0.05). Conclusion LPS can induce PC12 cell apoptosis and SIRT1 protein expression is inhibited,indicating that SIRT1 may take part in the apoptosis and play a protective role to PC12 cells.