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目的观察沙利度胺对人骨肉瘤细胞U2OS迁移和侵袭的作用,并初步探讨其作用机制。方法分别用不同浓度沙利度胺处理人骨肉瘤细U2OS,采用划痕实验检测沙利度胺对细胞迁移的影响,Transwell小室检测沙利度胺对细胞侵袭的影响,q RT-PCR检测不同浓度沙利度胺作用于细胞后VEGFA、HIF1α和OPN基因的变化,蛋白免疫印迹检测沙利度胺对细胞VEGF、HIF1和OPN蛋白的作用。结果 50、100、200μg/m L沙利度胺作用于U2OS后,划痕实验中沙利度胺组细胞迁移距离明显少于对照组,沙利度胺作用12 h,其迁移距离分别为24 h对照组的(62.36±12.41)%、(57.89±11.23)%和(49.47±6.90)%;作用24 h,其迁移距离分别为24 h对照组的(62.36±12.41)%、(57.89±11.23)%和(49.47±6.90)%。侵袭实验中显示沙利度胺组穿膜细胞数量对比对照组减少,其比率分别为(83.49±2.10)%、(70.64±2.86)%和(50.46±1.59)%。q RTPCR显示50、100、200μg/m L沙利度胺作用于U2OS 24 h后VEGFA、HIF-1α和OPN表达与对照组比较降低,VEGFA m RNA表达量分别为(2.50±0.25)%、(11.75±0.2)%和(6.51±0.01)%;HIF-1αm RNA表达量分别为(34.33±0.42)%、(46.33±1.06)%和(42.60±1.46)%;OPN表达量分别为(86.33±1.8)%、(52.07±1.29)%和(40.06±1)%。蛋白免疫印结果显示VEGFA、HIF-1α和OPN表达与对照组比较降低:与对照组VEGFA表达量1.06±0.06相比,药物组分别为0.88±0.01、04±0.02和0.62±0.01;与对照组HIF-1α表达量0.71±0.01对比,药物组分别为0.69±0.01、0.49±0.01和0.39±0.01;与对照组OPN表达量1.07±0.01对比,药物组分别为1.04±0.01、0.96±0.01和0.6±0.01。结论沙利度胺抑制骨肉瘤细胞U2OS迁移侵袭,其机制与降低细胞内VEGFA、HIF-1α和OPN有关。
Objective To observe the effect of thalidomide on the migration and invasion of U2OS in human osteosarcoma cells and to explore its mechanism. Methods Different concentrations of thalidomide were used to treat human osteosarcoma, and the effect of thalidomide on cell migration was detected by scratch test. The effect of thalidomide on cell invasion was detected by Transwell chamber. The effect of thalidomide on the expression of VEGF, HIF1αand OPN in cells was detected by western blot. Results After thalidomide treatment with 50, 100 and 200 μg / ml of thalidomide, the migrating distance of thalidomide group was significantly less than that of the control group after thalidomide treatment for 12 h and the migration distance was 24 (62.36 ± 12.41)%, (57.89 ± 11.23)% and (49.47 ± 6.90)% respectively in the control group and 62.36 ± 12.41% (57.89 ± 11.23) in the 24 h control group )% And (49.47 ± 6.90)%. The invasion assay showed that the number of transmembrane cells in thalidomide group decreased compared with the control group (83.49 ± 2.10), (70.64 ± 2.86)% and (50.46 ± 1.59)%, respectively. qRTPCR showed that the expression of VEGFA, HIF-1alpha and OPN in 50,100,200μg / ml L treated with thalidomide for 24 h decreased compared with the control group, the expression of VEGFA m RNA was (2.50 ± 0.25)%, ( The expression of HIF-1αmRNA was (34.33 ± 0.42)%, (46.33 ± 1.06)% and (42.60 ± 1.46)%, respectively. The expression levels of OPN were (86.33 ± 11.75 ± 0.2)% and 1.8%), (52.07 ± 1.29)% and (40.06 ± 1)%, respectively. Compared with the control group, the expression of VEGFA, HIF-1α and OPN in the protein immunosuppression results showed that compared with the control group, the expression of VEGFA was decreased by 1.06 ± 0.06 compared with the control group (0.88 ± 0.01,04 ± 0.02 and 0.62 ± 0.01, respectively) The expression of HIF-1α was 0.71 ± 0.01, the drug group was 0.69 ± 0.01, 0.49 ± 0.01 and 0.39 ± 0.01, respectively. Compared with the control group, the expression of OPN was 1.07 ± 0.01, the drug group was 1.04 ± 0.01,0.96 ± 0.01 and 0.6 ± 0.01. Conclusions Thalidomide inhibits migration and invasion of U2OS in osteosarcoma cells, and its mechanism is related to the decrease of intracellular VEGFA, HIF-1α and OPN.