SIRT3/MnSOD/ROS参与调控脂肪源性干细胞成骨分化的机制研究

Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats

目的:研究脂肪源性干细胞成骨分化过程中,SIRT3/MnSOD/ROS信号通路的调控作用。方法:提取SD大鼠脂肪源性干细胞(adipose derived stem cell,ADSC)并通过细胞表面标志物(CD29、CD44、CD34、CD45)及成骨、成脂多向分化鉴定。体外诱导其成骨分化,检测SIRT3表达情况;利用慢病毒转染抑制SIRT3表达,用茜素红染色检测ADSC成骨分化情况,qPCR检测成骨相关基因Runx2、ALP、OCN的表达,同时利用试剂盒检测锰超氧化物歧化酶(manganese superoxide dismutase,MnSOD)的活性及胞内活性氧簇(reactive oxygen species,ROS)水平。结果:ADSC成骨分化过程中,细胞内SIRT3 mRNA表达(7 d:1.71±0.03,15.21±0.17,P=0.014,n=5;14 d:1.44±0.04,9.70±0.18,P=0.002,n=5)及蛋白质表达(7 d:0.70±0.01,2.99±0.07,P=0.012,n=5;14 d:1.00±0.08,2.74±0.50,P=0.009,n=5)增加。转染SIRT3慢病毒可抑制SIRT3 mRNA表达(7 d:1.02±0.05,0.36±0.01,P=0.011,n=5;14 d:1.04±0.08,0.30±0.02,P=0.002,n=5)及蛋白质表达(7 d:0.88±0.04,0.29±0.01,P=0.034,n=5;14 d:1.18±0.03,0.43±0.01,P=0.014,n=5),导致ADSC成骨分化能力降低,MnSOD活性减弱(0.91±0.13,0.29±0.01,P=0.002,n=5),细胞内ROS水平升高(7 d:1.00±0.10,2.66±0.32,P=0.013,n=5;14 d:1.24±0.10,2.59±0.31,P=0.014,n=5),与成骨相关的相应靶基因Runx2、ALP、OCN表达下降(Runx2:1.07±0.02,0.19±0.01,P=0.001,n=5;ALP:1.09±0.02,0.53±0.01,P=0.001,n=5;OCN:0.95±0.02,0.33±0.01,P=0.004,n=5)。加入NAC预处理细胞,可逆转敲减SIRT3对ADSC成骨分化的抑制作用(ROS:2.63±0.20,1.07±0.19,P=0.010,n=5)。结论:ADSC成骨分化过程中,线粒体内SIRT3表达增加。SIRT3通过增强MnSOD的活性来降低胞内ROS水平,从而介导ADSC成骨分化。

Objective:To study the regulatory effect of SIRT3/MnSOD/ROS signaling pathway on osteogenic differentiation of adipose derived stem cells(ADSC). Methods:In this study,we first cultured ADSC isolated from rat inguinal fat pads,and ADSC were identified by surface markers(CD29,CD44,CD34,CD45)and they could be induced to osteogenic and adipogenic differentiation. During osteogenic differentiation induction in vitro,expressions of SIRT3 were studied by western blotting. We used Lenti-SIRT3 shRNA transfection to knockdown SIRT3 within ADSC,osteogenic capacities were identified by ARS staining,osteogenic differentiation-related genes Runx2,ALP,OCN mRNA expressions were assessed by qPCR,and the activity of MnSOD and reactive oxygen species(ROS)levels within ADSC were tested using kits. Results:The expression of SIRT3 m RNA(7 d:1.71±0.03,15.21±0.17,P=0.014,n=5;14 d:1.44±0.04,9.70±0.18,P=0.002,n=5)and protein(7 d:0.70±0.01,2.99±0.07,P=0.012,n=5;14 d:1.00±0.08,2.74±0.50,P =0.009,n =5)increased during osteogenic differentiation of ADSC. Lenti-SIRT3 sh RNA transfection could inhibit SIRT3 m RNA expression(7 d:1.02±0.05,0.36±0.01,P=0.011,n=5;14 d :1. 04 ± 0. 08,0. 30 ± 0. 02,P = 0. 002,n = 5) and protein expression(7 d:0.88±0.04,0.29±0.01,P=0.034,n=5;14 d:1.18±0.03,0.43±0.01,P=0.014,n=5),with decreased Mn SOD activ-ities(0.91±0.13,0.29±0.01,P=0.002,n=5)and increased ROS levels(7 d:1.00 ±0.10,2.66±0.32,P=0.013,n=5;14 d:1.24±0.10,2.59±0.31,P=0.014,n=5). The expression of corresponding target genes Runx2,ALP and OCN related to osteogenesis decreased(Runx2:1.07±0.02,0.19±0.01,P=0.001,n=5;ALP:1.09±0.02,0.53±0.01,P=0.001,n=5;OCN:0.95±0.02,0.33±0.01,P=0.004,n=5). Inhibition of osteogenic differentiation,induced by knockdown of SIRT3,could be reversed by pretreatment of NAC(ROS:2.63 ±0.20,1.07±0.19,P=0.010,n=5). Conclusion:SIRT3 within mitochondria plays vital roles in promoting osteogenic differentiation of ADSC,by mediating enhancement of Mn SOD activities and reduction of intracellular ROS levels.