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人黑色素瘤细胞A375是P15~(INK4b)缺失细胞系,通过细胞转染技术将外源P15~(INK4b)cDNA转染A375细胞,经PCR和Western blot检测,证明构建了P15~(INK4b)稳定表达细胞模型MLIK6。流式细胞光度术结果显示,与对照组MLC2相比,MLIK6细胞G_1期升高11％,S期下降14％。利用TdR-N_2O同步法所得同步的MLIK6和MLC2的M期细胞比例分别达到89.1％和76.8％,而G_1期的比例分别为74.3％和76.4％。~3H-TdR掺入的结果显示,MLIK6从G_1期进入S期的时间比MLC2延长2 h,并且掺入强度明显减弱。进一步探讨P15~(INK4b)对G_1/S相关调控基因的影响,发现G_1期的MLIK6细胞在诱导P27表达升高的同时,cyclinD1,CdK4,cyclinE和c-myc的蛋白水平均降低,其中cyclinD1的抑制在晚G_1期附近最显著,而对cyclinE的抑制则在G_1/S转换处表现突出,癌基因c-myc表达受到明显抑制并贯穿于G_1→S全部时间进程中。说明P15~(INK4b)是通过对G_1期不同阶段细胞周期引擎分子cyclinD1,CdK4,cyclinE和癌基因c-myc的抑制及增加CKI P27延缓了G_1/S进程。
The human melanoma cell line A375 is a P15~INK4b deletion cell line. The transfected P15~(INK4b) cDNA was transfected into A375 cells by cell transfection. PCR and Western blot analysis confirmed the construction of P15~INK4b stable. Expression cell model MLIK6. Flow cytometry results showed that compared with the control group MLC2, the MLIK6 cells increased by 11% in the G1 phase and decreased by 14% in the S phase. The proportion of M-phase cells of synchronized MLIK6 and MLC2 obtained by TdR-N 2 O synchronization method reached 89.1% and 76.8%, respectively, while the proportion of G 1 phase was 74.3% and 76.4%, respectively. The results of ~3H-TdR incorporation showed that the time for MLIK6 to enter the S phase from the G1 phase was 2 h longer than that of the MLC2, and the incorporation intensity was significantly reduced. To further investigate the effect of P15~(INK4b) on G1/S related regulatory genes, it was found that G1 phase MLIK6 cells induced increased expression of P27, while protein levels of cyclinD1, CdK4, cyclinE, and c-myc decreased, among them cyclinD1 Inhibition was most pronounced near the late G 1 phase, while inhibition of cyclin E was prominent at the G 1 /S transition, and the oncogene c-myc expression was significantly inhibited and permeated throughout the time course of G 1 →S. This indicated that P15~(INK4b) delayed the G1/S process by inhibiting cell cycle engine molecules cyclinD1, CdK4, cyclinE and c-myc in different phases of G1 phase and increasing CKI P27.