中华皮肤科杂志 ›› 2008, Vol. 41 ›› Issue (9): 598-600.

• 论著 • 上一篇    下一篇

BRAFV599E突变基因对A375细胞生长的影响

韩永智 孙建方 曾学思   

  1. 广州市广东省人民医院皮肤科 南京医科院皮研所 南京医科院皮研所
  • 收稿日期:2007-11-14 修回日期:2008-01-17 发布日期:2008-09-15
  • 通讯作者: 韩永智 E-mail:hyzderma@163.com

Mutated BRAFV599E gene enhances the growth of malignant melanoma cell line A375

  

  • Received:2007-11-14 Revised:2008-01-17 Published:2008-09-15

摘要: 目的 探讨BRAFV599E突变对黑素瘤生长的作用。方法 使用BRAF表达受到抑制的黑素瘤细胞Abraf1和Abraf2,以及BRAF表达未受抑制的A375细胞和Aneg细胞,MTT比色实验和平板克隆形成实验分别检测4种细胞的生长和克隆形成能力,流式细胞仪检测细胞周期和凋亡的变化。结果 与A375和Aneg细胞相比,Abraf1和Abraf2细胞的体外生长(F = 25.48,P < 0.001)和克隆形成(F = 90.06,P < 0.001)均受到明显抑制,S期细胞减少(F = 147.87,P < 0.001),G1期细胞增多(F = 9.14,P < 0.05),但细胞凋亡没有显著增加(F = 2.27,P > 0.05)。结论 BRAFV599E突变基因能促使黑素瘤细胞从G1期进入S期,从而增强黑素瘤细胞的体外生长和增殖能力,但对黑素瘤细胞的体外凋亡没有影响。

Abstract: Objective To investigate the role of mutated BRAFV599E gene in the growth of malignant melanoma cells. Methods In the previous study, plasmids containing small hairpin RNAs (shRNAs), braf1 and braf2 specific for mutated BRAFV599E gene, were designed and used to transfect A375 cells to inhibit the expression of BRAF gene in these cells. In this study, four kinds of A375 cells, including Abraf1 (transfected with braf1), Abraf2 (transfected with braf2), Aneg (transfected with negative plasmid) and A375 (untransfected) cells, were chosen and cultured in 96-well plate. MTT assay, plate clone forming assay, flow cytometry were applied to test the growth, clone formation, cell cycle and apoptosis of these cells respectively. Results Compared with A375 and Aneg cells, inhibited proliferation (F = 25.48, P < 0.001) and clone-forming rate (F = 90.06, P < 0.001) were observed in Abraf1 and Abraf2 cells; furthermore, flow cytometry showed a decrease in S-phase population(F = 147.87, P < 0.001) but an increase in G1-phase population(F = 9.14, P < 0.05)in Abraf1 and Abraf2 cells. However, neither Abraf1 nor Abraf2 cells exhibited a significant increase in apoptosis ratio(F = 2.27, P > 0.05). Conclusions Mutated BRAFV599E gene could induce the switch from G1 phase to S phase in melanoma cells, subsequently accelerate the growth of melanoma cells, but it has no obvious influence on the apoptosis of these cells.