鞘氨醇激酶1对人骨肉瘤U2OS细胞增殖、凋亡的影响及机制
发布时间：2019-07-12 18:44
【摘要】：目的探讨鞘氨醇激酶1(Sph K1)对人骨肉瘤U2OS细胞增殖和凋亡的影响及机制。方法将体外培养的人骨肉瘤U2OS细胞随机分为四组,转染组、Vector组分别转染Sph K1 siRNA及对照siRNA,PF-543组在培养液中加入1μmol/L Sph K1特异性抑制剂PF-543,对照组常规培养。干预48 h时,采用Western blotting法检测Sph K1蛋白表达,明确siRNA转染可在蛋白水平抑制骨肉瘤U2OS细胞Sph K1高表达,且效果与其抑制剂PF-543相当。采用MTT法检测各组细胞存活率,采用TUNEL法和流式细胞术检测细胞凋亡率,采用Western blotting法检测增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)、Bax、Bcl-2、Caspase-3及活化的Caspase-3(cleaved Caspase-3)蛋白表达。结果与对照组、Vector组比较,转染组、PF-543组细胞存活率均下降,细胞凋亡率均增加,PCNA、Cyclin D1、Bcl-2蛋白表达均减低,Bax、Caspase-3及cleaved Caspase-3蛋白表达均增加,组间比较P均0.05。结论 Sph K1基因可促进骨肉瘤细胞增殖、抑制细胞凋亡,调节凋亡及增殖相关因子表达可能是其作用机制。
[Abstract]:Objective to investigate the effect and mechanism of spannin kinase 1 (Sph K1) on proliferation and apoptosis of human osteosarcoma U2OS cells. Methods Human osteosarcoma U2OS cells cultured in vitro were randomly divided into four groups: Sph K1 siRNA in Vector group and PF-543, control group supplemented with 1 渭 mol / L Sph K1 specific inhibitor in the culture medium. After 48 hours of intervention, the expression of Sph K1 protein was detected by Western blotting assay. It was confirmed that siRNA transfection could inhibit the high expression of Sph K1 in osteosarcoma U2OS cells at the protein level, and the effect was similar to that of PF-543, an inhibitor of osteosarcoma. The cell survival rate was detected by MTT assay, the apoptosis rate was detected by TUNEL assay and flow cytometry, and the expression of proliferating cell nuclear antigen (PCNA), cell cycle protein D1 (Cyclin D1), Bax,Bcl-2,Caspase-3 and activated Caspase-3 (cleaved Caspase-3) protein was detected by Western blotting assay. Results compared with the control group and Vector group, the cell survival rate, apoptosis rate, PCNA,Cyclin D1, Bcl-2 protein expression, Bax,Caspase-3 and cleaved Caspase-3 protein expression in the transfer group and PF-543 group were decreased, while those in the PCNA,Cyclin D1 group and Bcl-2 group were decreased, while those in the Bax,Caspase-3 and cleaved Caspase-3 groups were all increased (P 0.05). Conclusion Sph K1 gene can promote the proliferation of osteosarcoma cells, inhibit apoptosis, regulate apoptosis and the expression of proliferation related factors may be its mechanism.
【作者单位】： 保定市莲池区妇幼保健院;保定市骨科医院;河北医科大学;
【基金】：河北省教育厅青年基金项目(QN2016018)
【分类号】：R738.1
[Abstract]:Objective to investigate the effect and mechanism of spannin kinase 1 (Sph K1) on proliferation and apoptosis of human osteosarcoma U2OS cells. Methods Human osteosarcoma U2OS cells cultured in vitro were randomly divided into four groups: Sph K1 siRNA in Vector group and PF-543, control group supplemented with 1 渭 mol / L Sph K1 specific inhibitor in the culture medium. After 48 hours of intervention, the expression of Sph K1 protein was detected by Western blotting assay. It was confirmed that siRNA transfection could inhibit the high expression of Sph K1 in osteosarcoma U2OS cells at the protein level, and the effect was similar to that of PF-543, an inhibitor of osteosarcoma. The cell survival rate was detected by MTT assay, the apoptosis rate was detected by TUNEL assay and flow cytometry, and the expression of proliferating cell nuclear antigen (PCNA), cell cycle protein D1 (Cyclin D1), Bax,Bcl-2,Caspase-3 and activated Caspase-3 (cleaved Caspase-3) protein was detected by Western blotting assay. Results compared with the control group and Vector group, the cell survival rate, apoptosis rate, PCNA,Cyclin D1, Bcl-2 protein expression, Bax,Caspase-3 and cleaved Caspase-3 protein expression in the transfer group and PF-543 group were decreased, while those in the PCNA,Cyclin D1 group and Bcl-2 group were decreased, while those in the Bax,Caspase-3 and cleaved Caspase-3 groups were all increased (P 0.05). Conclusion Sph K1 gene can promote the proliferation of osteosarcoma cells, inhibit apoptosis, regulate apoptosis and the expression of proliferation related factors may be its mechanism.
【作者单位】： 保定市莲池区妇幼保健院;保定市骨科医院;河北医科大学;
【基金】：河北省教育厅青年基金项目(QN2016018)
【分类号】：R738.1
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