中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (1): 10-13.

• 论著 • 上一篇    下一篇

HINT1对人黑素瘤细胞A375增殖和凋亡的影响

关杨1,姜祎群1,高莹2,陈浩1,曾学思1,孙建方1   

  1. 1. 南京 中国医学科学院北京协和医学院皮肤病研究所
    2. 南京医科院皮研所
  • 收稿日期:2009-02-23 修回日期:2009-04-17 出版日期:2010-01-15 发布日期:2010-01-05
  • 通讯作者: 关杨 E-mail:gygymi@gmail.com
  • 基金资助:

    中国医学科学院皮肤病研究所基金资助（303-05-7024）;不符合上述请自行填写具体基金资助项目名称（编号）

Effects of histidine triad nucleotide-binding protein 1 （HINT1） on the proliferation and apoptosis of human melanoma cell line A375

  • Received:2009-02-23 Revised:2009-04-17 Online:2010-01-15 Published:2010-01-05

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摘要：

目的 探讨HINT1基因对人黑素瘤细胞A375增殖、凋亡的影响及其作用机制。方法 应用构建的pcDNA.3.1/myc-His（-）A-HINT1真核表达载体，建立稳定转染且能高表达HINT1的A375细胞模型。用MTT法检测细胞增殖活性变化，流式细胞仪检测细胞周期及细胞凋亡率，TUNEL法检测细胞凋亡。分光光度法检测Caspase 3, Caspase 8 及Caspase 9的相对活性。Western印迹法检测bcl-2、bax、细胞色素C 及 p53的表达。 结果 与空载体-A375及未转染A375细胞相比，HINT1-A375细胞生长速度明显减慢（P < 0.05）；G1期细胞增多（73.17% ± 3.99%，F = 25.65，P < 0.05），S期细胞减少（16.75% ± 1.62%，F = 75.48，P < 0.01），细胞出现明显的晚期凋亡（23.57% ± 9.58%，F = 11.71，P < 0.01 ）。TUNEL法显示凋亡阳性细胞百分比（12% ± 1%）显著增高（F = 358.02，P < 0.01 ）；Caspase 9（0.45 ± 0.03，F = 135.62，P < 0.01）和Caspase 3表达（0.46 ± 0.04，F = 90.28，P < 0.01）升高。凋亡相关蛋白bax、细胞色素C 及 p53表达增加，bcl-2表达降低。结论 高表达HINT1可抑制A375细胞的增殖，促进其凋亡，细胞周期出现G1期阻滞，提示HINT1可能是人黑素瘤的抑癌基因。

关键词: 组氨酸三聚体核苷结合蛋白1, 黑色素瘤, 增殖, 凋亡

Abstract:

Objective To explore the effects of HINT1 on the proliferation and apoptosis of human melanoma cell line A375. Methods Three cell clones were used in the experiment, including A375 cells that were previously transfected with eukaryotic expressing vector pcDNA.3.1/myc-His（-）A-HINT1 and highly expressed HINT1 （HINT1-A375）, A375 cells transfected with pcDNA.3.1/myc-His（-）A empty vector （neo-A375） and untransfected A375 cells. MTT assay, flow cytometry and terminal deoxynucleotidyl transferase （TdT）-mediated dUTP nick end labeling （TUNEL） were performed to detect the proliferation, cell cycle and apoptosis of the three kinds of cells, respectively. The relative activity of Caspase 3, 8 and 9 was measured by spectrophotometry, and the protein expression of Bcl-2, Bax, Cytochrome C and p53 by Western blot. Results Compared with neo-A375 and untransfected A375 cells, HINT1-A375 cells grew more slowly （P < 0.05） with an increase in G1-phase population （73.17% ± 3.99%, F = 25.65, P < 0.05）, a decrease in S-phase population（16.75% ± 1.62%, F = 75.48, P < 0.01）, and a pronounced late apoptosis （23.57% ± 9.58%, F = 11.71, P < 0.01）. A significant increase was also observed in the percentage of apoptotic cells （12% ± 1%, F = 358.02, P < 0.01） as well as in the relative activity of Caspase 9 （0.45 ± 0.03, F = 135.62, P < 0.01） and Caspase 3 （0.46 ± 0.04, F = 90.28，P < 0.01） in HINT1-A375 cells compared with the other two kinds of cells. Western blot showed upregulated expressions of Bax, Cytochrome C and p53 but downregulated expression of Bcl-2 in HINT1-A375 cells. Conclusions The overexpression of HINT1 could inhibit the proliferation and promote the apoptosis of A375 cells with cell cycle arrest in G1 phase, hinting that HINT1 may be a tumor suppressor gene in human melanoma.

Key words: HINT1, Melanoma, Proliferation, Apoptosis