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目的:观测miR-17-5p在年轻的和衰老骨髓间充质干细胞(BMMSCs)中的表达,并分析其与细胞衰老的相关性。方法:采用密度梯度离心法分离大鼠BMMSCs并培养至第10代。取第4代(P4)BMMSCs和第10代(P10)BMMSCs的用细胞衰老β-半乳糖苷酶(β-gal)染色法进行染色。采用实时定量PCR检测P4及P10 BMMSCs中miR-17-5p基因的表达,并用2(-ΔΔct)法定量分析。结果:①形态学改变:P4～P10的细胞形态发生改变,P4的细胞形态均一,呈典型的漩涡状生长,P10的细胞失去典型的梭形外观,细胞体积增大而不规则,形状扁平破碎,细胞间隙增大,出现凋亡。②β-gal染色结果:P10的细胞肥大、染呈蓝色(呈阳性),P4的细胞染色后未着色(呈阴性)。③实时定量PCR检测结果:P4细胞miR-17-5p基因的表达量是P10细胞的6.5倍(P<0.01)。结论:衰老BMMSCs中miR-17-5p的表达量显著降低,提示miR-17-5p可能参与了细胞的衰老过程。
OBJECTIVE: To observe the expression of miR-17-5p in young and aged bone marrow mesenchymal stem cells (BMMSCs) and to analyze its correlation with cell senescence. Methods: Rat BMMSCs were isolated by density gradient centrifugation and cultured until passage 10. The 4th generation (P4) BMMSCs and the 10th generation (P10) BMMSCs were stained with the cell senescence β-galactosidase (β-gal) staining method. Real-time quantitative PCR was used to detect the expression of miR-17-5p gene in P4 and P10 BMMSCs, and was quantitatively analyzed by 2 (-ΔΔct) method. RESULTS: ① The morphological changes: the cell morphology of P4 ~ P10 changed. The cell morphology of P4 was homogeneous and showed a typical swirling growth. The cells of P10 lost the typical spindle shape appearance. The cell volume increased but irregularly and the shape was flat and broken , Cell gap increases, apoptosis occurs. ②β-gal staining results: P10 cells hypertrophy, stained blue (positive), P4 cells stained unstained (negative). ③ Real-time quantitative PCR results showed that the expression level of miR-17-5p gene in P4 cells was 6.5 times that of P10 cells (P <0.01). Conclusion: The expression of miR-17-5p in aging BMMSCs is significantly decreased, suggesting that miR-17-5p may be involved in the process of cell senescence.