【摘要】：目的探討鞘氨醇激酶1(Sph K1)對人骨肉瘤U2OS細(xì)胞增殖和凋亡的影響及機(jī)制。方法將體外培養(yǎng)的人骨肉瘤U2OS細(xì)胞隨機(jī)分為四組,轉(zhuǎn)染組、Vector組分別轉(zhuǎn)染Sph K1 siRNA及對照siRNA,PF-543組在培養(yǎng)液中加入1μmol/L Sph K1特異性抑制劑PF-543,對照組常規(guī)培養(yǎng)。干預(yù)48 h時(shí),采用Western blotting法檢測Sph K1蛋白表達(dá),明確siRNA轉(zhuǎn)染可在蛋白水平抑制骨肉瘤U2OS細(xì)胞Sph K1高表達(dá),且效果與其抑制劑PF-543相當(dāng)。采用MTT法檢測各組細(xì)胞存活率,采用TUNEL法和流式細(xì)胞術(shù)檢測細(xì)胞凋亡率,采用Western blotting法檢測增殖細(xì)胞核抗原(PCNA)、細(xì)胞周期蛋白D1(Cyclin D1)、Bax、Bcl-2、Caspase-3及活化的Caspase-3(cleaved Caspase-3)蛋白表達(dá)。結(jié)果與對照組、Vector組比較,轉(zhuǎn)染組、PF-543組細(xì)胞存活率均下降,細(xì)胞凋亡率均增加,PCNA、Cyclin D1、Bcl-2蛋白表達(dá)均減低,Bax、Caspase-3及cleaved Caspase-3蛋白表達(dá)均增加,組間比較P均0.05。結(jié)論 Sph K1基因可促進(jìn)骨肉瘤細(xì)胞增殖、抑制細(xì)胞凋亡,調(diào)節(jié)凋亡及增殖相關(guān)因子表達(dá)可能是其作用機(jī)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.
【作者單位】： 保定市蓮池區(qū)婦幼保健院;保定市骨科醫(yī)院;河北醫(yī)科大學(xué);
【基金】：河北省教育廳青年基金項(xiàng)目(QN2016018)
【分類號】：R738.1

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