CAJ | 학술논문

目的：探讨过氧化氢(H2 O2)对 NlH/3T3细胞中蛋白磷酸酶2A 催化亚基 C(PP2Ac)甲基化的影响。方法① H2 O20,0.1,1,5,10和25 mmol??L-1刺激 NlH/3T3细胞1 h,刃天青还原率检测细胞活性；② Western 蛋白印迹法检测 H2 O20,0.1,1和5 mmol??L-1作用1 h 后细胞内去甲基 PP2Ac 的表达；③分别用羧基端甲基酯酶(PME-1)抑制剂 AMZ300,0.1,0.5,1.0,5.0和10.0μmol??L-1和抗氧化剂 N-乙酰半胱氨酸(NAC)0,0.5,1和5 mmol??L-1预处理细胞30 min,再用 H2 O21 mmol??L-1作用细胞1 h,Western 蛋白印迹法检测细胞内去甲基 PP2Ac 的表达；④免疫荧光实验观察 NlH/3T3细胞 H2 O21 mmol??L-1处理1 h、AMZ3010.0μmol??L-1或 NAC 1 mmol??L-1分别预处理30 min 后再加 H2 O21 mmol??L-1处理1 h 细胞内去甲基 PP2Ac 的表达。结果①刃天青检测实验结果显示,H2 O2对细胞增殖具有显著的抑制作用,浓度≥0.1 mmol??L-1细胞活性显著降低(P<0.01)；② Western 蛋白印迹检测结果显示,H2 O2可致 NlH/3T3细胞内去甲基 PP2Ac 蛋白表达升高,H2 O20.1,1和5 mmol??L-1组与空白对照组相比差异有统计学意义(P<0.01)；③ Western 蛋白印迹结果显示,AMZ30与 NAC 二者均可拮抗 H2 O2对细胞内 PP2Ac 去甲基的作用,与H2 O21 mmol??L-1组相比,加入 AMZ305.0和10.0μmol??L-1或 NAC 1和5 mmol??L-1细胞内去甲基 PP2Ac含量明显降低(P<0.01)；④免疫荧光实验结果显示,加入 H2 O21 mmol??L-1刺激1 h 后,细胞内去甲基PP2Ac 的表达要高于正常细胞组,而用 AMZ3010.0μmol??L-1与 NAC 1 mmol??L-1干预后,去甲基 PP2Ac表达降低。结论 H2 O2可诱导 NlH/3T3细胞内 PP2Ac 去甲基,而 PME-1抑制剂 AMZ30和抗氧化剂NAC 均能拮抗 H2 O2诱导的 PP2Ac 去甲基作用。
목적：탐토과양화경(H2 O2)대 NlH/3T3세포중단백린산매2A 최화아기 C(PP2Ac)갑기화적영향。방법① H2 O20,0.1,1,5,10화25 mmol??L-1자격 NlH/3T3세포1 h,인천청환원솔검측세포활성；② Western 단백인적법검측 H2 O20,0.1,1화5 mmol??L-1작용1 h 후세포내거갑기 PP2Ac 적표체；③분별용최기단갑기지매(PME-1)억제제 AMZ300,0.1,0.5,1.0,5.0화10.0μmol??L-1화항양화제 N-을선반광안산(NAC)0,0.5,1화5 mmol??L-1예처리세포30 min,재용 H2 O21 mmol??L-1작용세포1 h,Western 단백인적법검측세포내거갑기 PP2Ac 적표체；④면역형광실험관찰 NlH/3T3세포 H2 O21 mmol??L-1처리1 h、AMZ3010.0μmol??L-1혹 NAC 1 mmol??L-1분별예처리30 min 후재가 H2 O21 mmol??L-1처리1 h 세포내거갑기 PP2Ac 적표체。결과①인천청검측실험결과현시,H2 O2대세포증식구유현저적억제작용,농도≥0.1 mmol??L-1세포활성현저강저(P<0.01)；② Western 단백인적검측결과현시,H2 O2가치 NlH/3T3세포내거갑기 PP2Ac 단백표체승고,H2 O20.1,1화5 mmol??L-1조여공백대조조상비차이유통계학의의(P<0.01)；③ Western 단백인적결과현시,AMZ30여 NAC 이자균가길항 H2 O2대세포내 PP2Ac 거갑기적작용,여H2 O21 mmol??L-1조상비,가입 AMZ305.0화10.0μmol??L-1혹 NAC 1화5 mmol??L-1세포내거갑기 PP2Ac함량명현강저(P<0.01)；④면역형광실험결과현시,가입 H2 O21 mmol??L-1자격1 h 후,세포내거갑기PP2Ac 적표체요고우정상세포조,이용 AMZ3010.0μmol??L-1여 NAC 1 mmol??L-1간예후,거갑기 PP2Ac표체강저。결론 H2 O2가유도 NlH/3T3세포내 PP2Ac 거갑기,이 PME-1억제제 AMZ30화항양화제NAC 균능길항 H2 O2유도적 PP2Ac 거갑기작용。
OBJECTIVE To investigate the effect of hydrogen peroxide(H2 O2 ) on protein phospha-tase 2A catalytic subunit C(PP2Ac) methylation. METHODS After H2O2 0, 0.1, 1, 5, 10 and 25 mmol.L-1 treatment of NlH/ 3T3 cells for 1 h, the rate of resazurin reduction was observed to determine the cell proliferation while Western blotting was used to detect the expression of un-methylated PP2Ac. PP2A methylesterase (PME-1) inhibitor AMZ30 0, 0.1, 0.5, 1, 5 and 10 μmol.L-1 and antioxidant NAC 0, 0.5, 1 and 5 mmol.L-1 were used to intervene in the effect of H2 O2 1 mmol.L-1 on NlH/ 3T3 cells. After various treatments, the expression of un-methylated PP2Ac was detected by Western blotting and indirect immunofluorescence. RESULTS Compared with normal control group, H2 O2 0.1, 1, 5 and 25 mmol.L-1 significantly decreased the cell proliferation ( P < 0.01). The expression of un-methylated PP2Ac in NlH/ 3T3 cells and in H2 O2 0.1, 1 and 5 mmol.L-1 groups was significantly increased(P<0.01) compared with normal control group. Western blotting results revealed that both NAC (1 or 5 mmol.L-1 ) and AMZ30 (5 or 10 μmol.L-1 ) significantly antagonized the effect of H2 O2 on PP2Ac(P<0.01). lndirect immunofluorescence results showed that the un-methylated PP2Ac expression of H2 O2 1 mmol.L-1 group was higher than that of normal control group, while the expression of PP2Ac methylation was reduced after the use of AMZ30 10 μmol.L-1 or NAC 1 mmol.L-1. CONCLUSION H2 O2 can induce demethylation of PP2Ac in NlH/ 3T3 cells, while the PME-1 inhibitor AMZ30 and antioxidant NAC can antagonize the methylation of PP2Ac induced by H2 O2 .