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目的研究小干扰RNA(siRNA)沉默A375黑素瘤细胞肾上腺髓质素(ADM)基因对其细胞周期的影响。方法利用人工合成的ADM靶向小分子干扰RNA转染A375细胞,采用实时荧光定量-PCR(qRT-PCR)筛选出有效的siRNA,用选出的干扰效果最好的siRNA转染的A375细胞为实验组;转染非特异序列的细胞为阴性对照组;未转染的细胞为空白对照组。流式细胞仪(FCM)检测干扰ADM基因48 h后A375细胞周期的情况。结果 FCM结果显示,siRNA干扰组G2/M期细胞比率为(11.360±1.224)%,明显高于未转染组的(1.020±0.990)%和非特异序列转染组的(4.150±1.032)%,差异有统计学意义(P<0.05);未转染组与非特异序列转染组差异无统计学意义(P>0.05)。而siRNA干扰组G1/G0、S期细胞比率分别为(69.443±2.023)%、(19.197±0.890)%,与未转染组(68.567±3.653)%、(30.417±4.582)%和非特异序列转染组(70.530±3.008)%、(23.313±4.645)%比较,差异均无显著性(P>0.05)。结论 ADM能够将黑素瘤细胞阻滞在G2/M期,抑制细胞增殖。
Objective To investigate the effect of small interfering RNA (siRNA) silencing the adrenomedullin (ADM) gene on the cell cycle of A375 melanoma cells. Methods Transfected A375 cells with artificially synthesized ADM-targeted small interfering RNAs and screened for effective siRNA using real-time fluorescence quantitative PCR (qRT-PCR). The selected siRNAs transfected with siRNA with the best interference effect were A375 cells. The experimental group; cells transfected with non-specific sequences were negative control groups; untransfected cells were blank control groups. Flow cytometry (FCM) was used to detect the A375 cell cycle after 48 h of interference with ADM gene. Results FCM results showed that the percentage of G2/M phase cells in the siRNA interference group was (11.360±1.224)%, which was significantly higher than that in the untransfected group (1.020±0.990)% and (4.150±1.032)% in the non-specific sequence transfection group. The difference was statistically significant (P<0.05); there was no significant difference between the non-transfected group and the non-specific sequence transfection group (P>0.05). The ratio of G1/G0 and S phase cells in the siRNA interference group was (69.443±2.023)%, (19.197±0.890)%, and (68.567±3.653)%, (30.417±4.582)% and non-specific sequences in the untransfected group, respectively. There was no significant difference (70.530±3.008)% and (23.313±4.645)% in transfection group (P>0.05). Conclusion ADM can block melanoma cells in G2/M phase and inhibit cell proliferation.