摘要
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(10μmol/L MG132作用4 h)和P53抑制剂pifithrin-α(PFT-α)+MG132组(20μmol/L PFT-α预干预1 h,随后加入10μmol/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。
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. Ear- ly -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 fur- ther 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 ceils. Application of the proteasome inhibitor MG132 reduced the proliferation of earl）, - 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 - pas- sage 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 ac- tivates P53 to suppress cell proliferation through blocking cell cycle progression.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2012年第12期2250-2253,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30973094)
山西省回国留学人员科研资助项目(No.2009-50)
关键词
蛋白酶体
P53
人骨髓间充质干细胞
细胞增殖
Proteasome
P53
Human bone marrow mesenchymal stem cells
Cell proliferation
