miR-141对股骨头坏死骨髓间质干细胞活性及VEGF/TGF-β_(2)蛋白的影响

Effects of miR-141 on Activity of Bone Marrow Mesenchymal Stem Cells and VEGF/TGF-β_(2) Protein in Femoral Head Necrosis

目的:探讨miR-141对股骨头坏死骨髓间质干细胞活性及VEGF/TGF-β_(2)蛋白的影响。方法:从我院收治的股骨骨折及激素性股骨头坏死患者手术期间抽取骨髓,培养及鉴定MSCs后随机分为四组,分别为A组(股骨颈骨折患者hMSCs细胞)、B组(激素性股骨头坏死患者hMSCs细胞)、C组(B组+miR-141-siRNA-NC)、D组(B组+miR-141-siRNA),MTT检测细胞活性;流式细胞仪检测凋亡率;免疫印迹及RT-PCR分别检测VEGF、TGF-β_(2)蛋白及mRNA水平;荧光酶素实验证明miR-141对VEGF、TGF-β_(2)调控作用。结果:与A组相比,B组hMSCs细胞24h、48h及72h活性降低(P<0.05),B组和C组24h、48h及72h hMSCs细胞OD值无意义(P>0.05),与C组相比,D组MSCs细胞24h、48h及72h活性升高,组间比较存在差异(P<0.05);A组、B组、C组及D组细胞凋亡率分别为(4.23±0.33)、(20.73±1.20)、(19.43±1.16)及(14.76±0.80),组间比较存在差异(F=381.00,P<0.001);与A组相比,B组VEGF、TGF-β_(2)表达降低(P<0.05),B组和C组VEGF、TGF-β_(2)表达无意义(P>0.05),与C组相比,D组VEGF、TGF-β_(2)表达升高,组间比较存在差异(P<0.05);荧光素酶实验证实VEGF、TGF-β_(2)是miR-141靶基因,当抑制miR-141时VEGF、TGF-β_(2)表达升高。结论:沉默miR-141能够通过提高股骨头坏死骨髓间质干细胞活性,抑制凋亡发挥髓间质干细胞保护作用,研究机制可能与激活VEGF、TGF-β_(2)活性相关。

Objective:To investigate the effects of Mir-141 on the activity of bone marrow mesenchymal stem cells and VEGF/TGF-β_(2) protein in femoral head necrosis.Methods:Patients with femoral fracture and steroid-induced femoral head necrosis admitted to our hospital were randomly divided into 4 groups after bone marrow extraction,culture and identification of MSCs during surgery,including group A(hMSCs cells from patients with femoral neck fracture),group B(hMSCs cells from patients with steroid-induced femoral head necrosis),group C(group B+Mir-141-sirNA-NC),and group D(group B+Mir-141-SiRNA).MTT was used to detect cell activity.The apoptosis rate was detected by flow cytometry.The protein and mRNA levels of VEGF and TGF-β_(2) were detected by western blot and RT-PCR,respectively.Luciferase assay demonstrated the regulatory effect of Mir-141 on VEGF and TGF-β_(2).Results:Compared with group A,the activity of hMSCs cells in group B was decreased at 24h,48h and 72h(P<0.05),while the OD values of hMSCs cells in groups B and C were insignificant at 24h,48h and 72h(P>0.05).Compared with group C,the activity of MSCs cells in group D was increased at 24h,48h and 72h.There was significant difference between groups(P<0.05).The apoptosis rates of group A,B,C and D were(4.23±0.33),(20.73±1.20),(19.43±1.16)and(14.76±0.80),respectively,and there were differences among groups(F=381.00,P<0.001).Compared with group A,the expressions of VEGF and TGF-β_(2) in group B were decreased(P<0.05),and the expressions of VEGF and TGF-β_(2) in groups B and C were insignificant(P>0.05).Compared with group C,the expressions of VEGF and TGF-β_(2) in group D were increased,and there were differences among groups(P<0.05).Luciferase assay confirmed that VEGF and TGF-β_(2) were the target genes of Mir-141,and the expressions of VEGF and TGF-β_(2) increased when Mir-141 was inhibited.Conclusion:The silencing of Mir-141 can play a protective role of mesenchymal stem cells by improving the activity of bone marrow mesenchymal stem cells in femur head necrosis and inhibiting apoptosis.The mechanism may be related to the activation of VEGF and TGF-β_(2) activities.