叔丁基对苯二酚激活骨髓间充质干细胞蛋白酶体活性延缓复制性衰老

目的:探讨叔丁基对苯二酚(tert-butylhydroquinone,t BHQ)在延缓骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)复制性衰老进程中的作用,以期为细胞移植治疗提供数量充足的种子细胞。方法:30μmol/L t BHQ持续作用于体外传代晚期BMSCs 4周,化学发光法测量蛋白酶体活性;CCK-8法检测细胞活力;Brd U掺入实验检测细胞增殖能力;流式细胞术检测细胞周期分布;衰老相关β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色检测衰老细胞百分比;Western blot检测衰老相关蛋白P53表达变化。结果:30μmol/L t BHQ持续作用于传代晚期BMSCs 4周后,蛋白酶体活性较DMSO溶剂对照组上调(21.96±1.98)%(P0.05)。CCK-8法显示随t BHQ浓度增加,细胞活力逐渐升高,至40μmol/L达到平台期,且至120μmol/L未见明显细胞毒性。Brd U掺入实验显示t BHQ组阳性细胞率与DMSO溶剂对照组比较,细胞增殖能力显著提高(P0.05);t BHQ组细胞增殖指数也显著高于DMSO组(P0.05)。t BHQ组SA-β-Gal阳性率较DMSO溶剂对照组显著降低(P0.01),且衰老相关蛋白P53表达量也较对照组下降(P0.05)。结论:t BHQ能够通过提高蛋白酶体活性延缓因蛋白酶体功能障碍引起的BMSCs复制性衰老进程。

tBHQ delayed replicative senescence by activating the proteasome system of BMSCs

AIM: To investigate the effect of tert-butylhydroquinone(tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells(BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately. The proteasomal activity was determined by chemiluminescence method. The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation. Percentage of senescent cells was detected by senescence-associated β-galactosidase(SA-β-Gal) staining. The expression of P53 was measured by Western blot.RESULTS: After the continuous treatment of tBHQ(30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05). The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed. BrdU-positive cells, which represented the cell proliferation, were significantly increased(P<0.05). The proliferation index was also significantly increased by flow cytometry analysis(P<0.05). The SA-β-Gal positive cells and the expression of P53 were decreased(P<0.05).CONCLUSION: tBHQ delays the proteasome dysfunction associated senescence progress of BMSCs by increasing the proteasomal activity.