镉致人支气管上皮细胞恶性转化中细胞周期的变化
Changes in Cell Cycle Distribution during Cadmium Induced Malignant Transformation of Human Bronchial Epithelial Cells
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摘要:
目的 探讨镉致人支气管上皮(16HBE)细胞恶性转化中细胞周期的变化,分析去甲基化处理后细胞周期蛋白基因(CyclinD1)的表达情况。 方法 采用流式细胞术检测正常16HBE细胞和氯化镉恶性转化16HBE细胞不同阶段(第5、15、35代细胞)及镉恶性转化第35代细胞接种裸鼠成瘤细胞(简称成瘤细胞)的细胞周期变化情况;采用实时荧光定量聚合酶链反应和Western blot法检测上述细胞及经去甲基化转移酶抑制剂5-氮杂-2-脱氧胞苷(5-Aza-dC)处理后的成瘤细胞CyclinD1的mRNA和蛋白表达情况。 结果 与正常16HBE细胞比较,氯化镉恶性转化16HBE细胞系第35代细胞及成瘤细胞在G0/G1期的细胞比例随着转化代数的增加而增大,分别为60.04%、64.12%;而在S期和G2/M期细胞比例则随着转化代数的增加而减少——在S期细胞比例分别为35.03%、31.83%,在G2/M期分别为4.93%、4.01%——差异均有统计学意义(均P < 0.05)。与正常16HBE细胞相比,氯化镉恶性转化16HBE细胞不同阶段(第5、15、35代细胞及成瘤细胞)的CyclinD1的mRNA和蛋白表达量呈现随着代数增加而上调的趋势;其中第35代细胞(2.34倍和2.41倍)及成瘤细胞(2.33倍和2.35倍)明显增加(均P < 0.05);5-Aza-dC处理后,成瘤细胞的CyclinD1的mRNA和蛋白表达增加量有所下降,分别为处理前16HBE细胞的1.815倍和1.742倍(P < 0.05)。 结论 镉恶性转化16HBE细胞中细胞周期发生变化,而这些改变可能与DNA甲基化相关。 Abstract:Objective To describe the changes in cell cycle distribution during malignant transformation of human bronchial epithelial cells (16HBE) induced by cadmium chloride treatment,and to assess the relationshiP between methylation and expression of CyclinD1. Methods Flow cytometry was used to detect the cell cycle distribution of normal 16HBE cells (control),cadmium chloride treated 16HBE cells at 5th,15th,and 35th passages,and tumorigenic cells from a nude mice model stimulated with the 35th passage 16HBE.Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) and Western blot were used to measure the expression levels of CyclinD1 mRNA and protein in the above cells and the tumorigenic cells treated with 5-aza-2-deoxycytidine. Results As cell passages increased,the proportions of the cadmium transformed cells and the tumorigenic cells increased in the G0/G1 phase (60.04% and 64.12% respectively),and decreased in the S (35.03% and 31.83% respectively) and G2/M phases (4.93% and 4.01% respectively),compared with the control cells (P < 0.05).The expression levels of CyclinD1 mRNA and protein also demonstrated increasing trends with the CdCl2-treated cell passages increased,and were significantly increased in the 35th passage-transformed cells (2.34-and 2.41-fold respectively) and the tumorigenic cells (2.33-and 2.35-fold respectively) compared with the controls (P < 0.05).However,the expression levels of CyclinD1 mRNA and protein of 5-Aza-2-deoxycytidine treated tumorigenic cells were decreased to 1.815-and 1.742-fold respectively of the 16HBE cells before the 5-Aza-2-deoxycytidine treatment. Conclusion The variations in cell cycle distribution of 16HBE cells induced by cadmium may be associated with DNA methylation.
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