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目的研究沙利度胺对人慢性粒细胞白血病急变株K562细胞凋亡及血管内皮生长因子(vascularendothelia growth factor,VEGF)分泌的影响。方法采用不同浓度的沙立度胺(0.5、1.0、2.0 mmol/L)作用于K562细胞24、48、72、96 h,瑞-姬(Wright-Giemsa)染色法观察细胞形态;四甲基偶氮唑盐(methylthiazolyl tetrozolium,MTT)法检测细胞增殖;流式细胞仪膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法检测凋亡率;琼脂糖凝胶电泳法检测脱氧核糖核酸梯状条带;酶联免疫吸附法检测VEGF浓度。结果培养24、48 h后,沙立度胺对K562细胞生长无抑制作用;作用72 h后,1.0、2.0 mmol/L浓度组开始出现对K562细胞生长的抑制(P<0.001);作用96 h后,0.5 mmol/L浓度组也产生对K562细胞生长的抑制(P<0.001),呈一定的浓度和时间依赖性。沙立度胺处理72 h后,K562细胞出现形态学改变,其体积缩小,出现空泡化,边缘出现突起,染色质浓缩、边集,核固缩、出现凋亡小体。经沙立度胺处理后,流式分析结果显示K562细胞凋亡率增加(P<0.001)。沙立度胺作用72 h后,琼脂糖凝胶电泳可见典型的DNA梯状条带。K562细胞培养48 h后,沙立度胺抑制VEGF的分泌(P<0.001),并且VEGF浓度与凋亡率呈负相关(r=-0.789)。结论沙利度胺抑制K562细胞的增殖,呈一定的浓度和时间依赖性;沙利度胺对K562细胞凋亡有明显诱导作用;沙利度胺抑制K562细胞VEGF的分泌。
Objective To investigate the effects of thalidomide on the apoptosis of human acute myeloid leukemia K562 cells and the secretion of vascular endothelial growth factor (VEGF). Methods K562 cells were treated with different concentrations of thalidomide (0.5, 1.0 and 2.0 mmol / L) for 24, 48, 72 and 96 h, respectively. Wright-Giemsa staining was used to observe the cell morphology. Cell proliferation was detected by methyl thiazolyl tetrozolium (MTT) method. Flow cytometry was used to detect apoptosis rate by Annexin V-FITC / propidium iodide double staining method. Deoxygenation was detected by agarose gel electrophoresis The ladder of ribosomal DNA was detected by enzyme linked immunosorbent assay. Results Thalidomide did not inhibit the growth of K562 cells 24 h and 48 h after incubation, and inhibited the growth of K562 cells at 1.0 and 2.0 mmol / L for 72 h (P <0.001) After 0.5 mmol / L, K562 cells also inhibited the growth of K562 cells (P <0.001) in a concentration- and time-dependent manner. After thalidomide treatment for 72 h, the morphology of K562 cells was changed. The volume of K562 cells was reduced, vacuolization was observed, the edge of the K562 cells appeared to protrude, the chromatin condensed, the edge set and nuclear pyknosis appeared, and apoptotic bodies appeared. After thalidomide treatment, the results of flow cytometry showed that the apoptosis rate of K562 cells increased (P <0.001). Thalidomide 72 hours after the agarose gel electrophoresis showed a typical DNA ladder. Thalidomide inhibited the secretion of VEGF (P <0.001) 48 h after K562 cells were cultured, and the concentration of VEGF was negatively correlated with the apoptosis rate (r = -0.789). Conclusion Thalidomide can inhibit the proliferation of K562 cells in a concentration-dependent and time-dependent manner. Thalidomide induces the apoptosis of K562 cells. Thalidomide inhibits the secretion of VEGF in K562 cells.