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目的探讨低浓度力达霉素(LDM)对人白血病细胞K562增殖的抑制作用及其可能的作用机制。方法以不同浓度LDM处理K562细胞48 h,通过MTT法检测LDM对细胞增殖的抑制作用;利用苔盼蓝染色对细胞活性进行测定;利用瑞氏-姬姆萨染色法观察LDM对细胞形态的影响;Elisa法检测肿瘤坏死因子-α(TNF-α)在细胞培养上清中的表达;采用Western Blot检测凋亡相关蛋白TNF-α的表达。结果 LDM对K562细胞增殖有一定的抑制作用,用10-12、10-11、10-10、10-9、10-8mol/L浓度的LDM分别作用K562细胞48 h,抑制率分别为(21.2±2.48)%、(37.5±0.88)%、(52.1±1.24)%、(64.5±1.61)%和(71.3±2.00)%。通过瑞氏姬姆萨染色,荧光显微镜下可见胞膜皱缩,有泡状突起及不规则凹陷,胞核染色质浓缩,固缩,浓染,断裂成团块分散在胞膜周边。酶联免疫吸附试验(ELISA)检测细胞培养上清,LDM处理组与对照组相比,TNF-α表达水平升高。Western Blot分析显示,分别用10-8、10-10、10-12 mol/L浓度的LDM处理细胞48 h后,TNF-α蛋白的表达量依次为0.42±0.01、1.67±0.13(P<0.01)和2.41±0.08(P<0.01),与对照组0.23±0.02相比,吸光度值比升高。结论低浓度LDM可能通过上调TNF-α表达水平诱导K562细胞凋亡抑制其增殖。
Objective To investigate the inhibitory effect of low concentration of lidamycin (LDM) on the proliferation of human leukemia cell K562 and its possible mechanism. Methods K562 cells were treated with different concentrations of LDM for 48 h. The inhibitory effect of LDM on cell proliferation was detected by MTT assay. Cell viability was determined by trypan blue staining. The effect of LDM on cell morphology was observed by Wright-Giemsa staining The expression of tumor necrosis factor-α (TNF-α) in cell culture supernatant was detected by Elisa method. The expression of TNF-α was detected by Western Blot. Results LDM could inhibit the proliferation of K562 cells. K562 cells were treated with 10-12, 10-11, 10-10, 10-9, and 10-8mol / L LDM for 48 h, respectively. The inhibition rates were (21.2 ± 2.48%, (37.5 ± 0.88)%, (52.1 ± 1.24)%, (64.5 ± 1.61)% and (71.3 ± 2.00)%, respectively. By Wright’s Giemsa staining, under the fluorescence microscope, cell membrane shrinkage, bubble-shaped protrusions and irregular pits, nuclear chromatin condensation, condensation, staining, broken into clumps around the cell membrane. Enzyme-linked immunosorbent assay (ELISA) detection of cell culture supernatant, LDM treatment group compared with the control group, TNF-α expression increased. Western Blot analysis showed that the expression of TNF-α protein was 0.42 ± 0.01,1.67 ± 0.13 (P <0.01) in the order of 10-8,10-10,10-12 mol / L LDM for 48 h ) And 2.41 ± 0.08 (P <0.01), and the ratio of absorbance increased compared with that of the control group (0.23 ± 0.02). Conclusion Low concentration of LDM may induce apoptosis of K562 cells by up-regulating the expression of TNF-α.