增龄条件下Wnt/β-catenin信号通路在骨髓间充质干细胞中的变化

摘要

目的:研究增龄条件下骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的增龄性改变及Wnt/β-catenin信号通路在增龄过程中的变化.方法:选用3月龄及18月龄的SD大鼠,双能X线检测股骨密度;分离培养BMSCs,SA-β-Gal染色分析3月龄组及18月龄组细胞衰老情况;实时定量PCR技术检测端粒酶(telomerase reverse transcriptase,TERT)mRNA表达;采用免疫酶联吸附试验检测肿瘤坏死因子(TNF-α)及白介素6(IL-6)的分泌情况;Western Blot法检测Wnt信号通路核心蛋白β-catenin在细胞浆及细胞核中的表达;实时定量PCR检测Wnt通路关键基因LEF-1、TCF-4的表达.结果:18月龄组大鼠股骨密度显著低于3月龄组;SA-β-Gal阳性表达细胞随年龄增加而增加,TERT表达水平随年龄增加而显著降低;18月龄组BMSCs TNF-α及IL-6的分泌量显著高于3月龄BMSCs;18月龄组BMSCs细胞核及细胞浆中β-catenin表达量较3月龄BMSCs均呈增高趋势;18月龄组BMSCs LEF-1的基因表达水平较3月龄BMSCs上升1.6倍,TCF-4的基因表达水平也上升3.12倍.结论:随着年龄的增加BMSCs分泌的炎症因子含量增加,导致Wnt/β-catenin信号通路活化进而影响干细胞的生物学功能.%Objective:To investigate the changes of bone marrow mesenchymal stem cells (BMSCs) and the changes of Wnt/β-catenin signaling pathway under aging condtion.Methods:3-month-old and 18-month-old were used in our study,the femur density were detect by dual energy X-ray.We isolated BMSCs from bone marrow and then the SA-β-Gal expression was analyzed to assess the cell senescence.The expression of telomerase reverse transcriptase (TERT) mRNA was detected by real-time quantitative PCR.The secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was detected by immuno-enzyme-linked immunosorbent assay.Western blot was used to detect the expression of β-catenin in the cytoplasm and nucleus.Real-time quantitative PCR was used to detect the expression of LEF-1 and TCF-4.Results:The femur density in the 18-month-old group was significantly lower than that in the 3-month-old group.The expression of SA-β-Gal positive cells increased in BMSCs (18-month-old group) compared with 3-month-old group,however,the expression of TERT was significantly decreased in BMSCs (18-month-old group).The secretion of TNF-α and IL-6 in BMSCs was significantly increased in 18-month-old group than in 3-month-old group.The expression of β-catenin in the nucleus and cytoplasm of BMSCs in 18-month-old group was higher than that in 3-month-old BMSCs group.The expression level of group LEF-1 in the 18-month-old group was 1.6 times higher than that of the 3-month-old group,and the gene expression level of TCF-4 also increased 3.12 times than that of the 3-month-old group.Conclusion:With the increase of age,the content of inflammatory cytokines secreted by BMSCs were increased,which led to the activation of Wnt/β-catenin signaling pathway and then affect the biological function of BMSCs.