蛋白酶体功能障碍通过激活P53抑制人骨髓间充质干细胞增殖

 目的：明确蛋白酶体活性下降导致人骨髓间充质干细胞（hBMSCs）增殖能力降低的机制，探讨P53在此过程中的调控作用，以期为组织工程提供数量充足且活性优良的种子细胞。方法：检测早期（第3~4代）与晚期（≥14代） hBMSCs蛋白酶体活性及P53表达变化。将早期hBMSCs分为DMSO对照组、蛋白酶体抑制剂MG132组（10 μmol/L MG132作用4 h）和P53抑制剂pifithrin-α（PFT-α）+ MG132组（20 μmol/L PFT-α预干预1 h，随后加入10 μmol/L MG132作用4 h），通过BrdU掺入实验和流式细胞术观察细胞增殖能力及细胞周期分布。随后将PFT-α直接作用于晚期hBMSCs，通过BrdU掺入实验检测P53抑制剂对晚期细胞增殖能力的影响。结果：体外培养晚期hBMSCs伴随蛋白酶体活性降低，P53表达水平较早期细胞上调16.89%±4.44%（P<0.05）。给予MG132能够显著抑制hBMSCs增殖，BrdU阳性率降低至33.36%±2.24%（P<0.01）；但是若提前给予PFT-α则能部分拮抗MG132对hBMSCs增殖能力的影响，BrdU阳性率上升至49.23%±2.67%（P<0.05）。流式细胞术结果显示，PFT-α+ MG132组各期细胞分布与DMSO对照组相似，增殖指数显著高于MG132组（P<0.01）。抑制P53激活能够显著提高晚期hBMSCs增殖能力，BrdU阳性率较对照组升高24.73%±8.35%（P<0.01）。结论：传代晚期hBMSCs蛋白酶体活性下降可能通过激活P53、影响细胞周期进程进而导致hBMSCs增殖潜能下调。

Dysfunction of proteasome inhibits the proliferation of hBMSCs via activation of P53

AIM: To investigate the role of P53 in decreased cell proliferation and proteasomal activity during culture expansion of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: The proteasomal activity and expression level of P53 in early-passage (passage 3~4) and late-passage (passage≥14) hBMSCs were observed. Early-passage hBMSCs were divided into DMSO control group, MG132 group (treated with 10 μmol/L MG132 for 4 h) and pifithrin-α(PET-α) + MG132 group (pretreatment with 20  μmol/L PFT-α for 1 h then exposure to MG132 for 4 h). The proliferation and cell cycle distribution of the cells were measured by BrdU incorporation assay and PI staining. To further confirm the effect of P53 inhibitor on late-passage hBMSCs, the cells were incubated with 20  μmol/L PFT-α and the BrdU-positive cells were counted. RESULTS: Accompanied by reduced proteasomal activity, the expression level of P53 in late-passage hBMSCs was up-regulated by 16.89%±4.44% compared with early-passage cells. Application of the proteasome inhibitor MG132 reduced the proliferation of early-passage hBMSCs, and the percentage of BrdU-positive cells dropped to 33.36%±2.24%. However, MG132-induced decrease in cell proliferation was partially reversed by pretreatment with PFT-α. BrdU-positive cells in PFT-α + MG132 group were increased to 49.23%±2.67%. The cell cycle distribution had no significant difference between PFT-α + MG132 group and DMSO group, and the higher proliferation index was found in PFT-α + MG132 group but not in MG132 group. Inhibition of P53 activity in late-passage hBMSCs by PFT-α promoted the cell proliferation, and the percentage of BrdU-positive cells was higher than that in control group. CONCLUSION: Long-term expansion of hBMSCs in vitro reduces proteasomal activity, which in turn activates P53 to suppress cell proliferation through blocking cell cycle progression.