中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (2): 68-70.

• 论著 • 上一篇    下一篇

TRP-1编码基因反义核酸对黑素细胞增殖及功能的影响

李春英, 高天文, 李廷慧, 孙东杰, 李强, 高鸽, 刘玉峰   

  1. 第四军医大学西京医院全军皮肤性病中心 西安 710032
  • 收稿日期:2003-03-06 出版日期:2004-02-15 发布日期:2004-02-15
  • 通讯作者: 高天文,E-mail:gaotw@fmmu.edu.cn E-mail:gaotw@fmmu.edu.cn

The Effect of Antisense TRP-1 on Proliferation and Functions of Melanocytes and Malignant Melanoma Cells

LI Chun-ying, GAO Tian-wen, LI Ting-hui, SUN Dong-jie, LI Qiang, GAO Ge, LIU Yu-feng   

  1. Centre of Dermatology of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China
  • Received:2003-03-06 Online:2004-02-15 Published:2004-02-15

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摘要： 目的 构建酪氨酸相关蛋白-1(TRP-1)反义真核表达载体并转染TRP-1高表达的黑素细胞及恶性黑素瘤细胞,进一步研究其对黑素细胞及恶性黑素瘤细胞增殖及功能的影响。方法 将TRP-1基因反向亚克隆入真核表达载体pcDNA3.1,转染黑素细胞及恶性黑素瘤细胞。以逆转录聚合酶链反应(RT-PCR)检测TRP-1mRNA水平的变化,免疫印迹法检测TRP-1蛋白水平的改变。通过流式细胞仪检测细胞周期的变化,并用L-多巴测定酪氨酸酶活性的改变。结果 成功构建重组反义表达载体pcDNA3.1/TRP-1(-),转染细胞稳定表达TRP-1反义核酸。RT-PCR提示TRP-1mRNA水平显著下降,免疫印迹提示其TRP-1蛋白表达水平降低。流式细胞检测表明反义TRP-1转染细胞发生细胞周期G1期阻滞。转染组黑素细胞及黑素瘤细胞酪氨酸酶活性抑制率分别达46%和54%。结论 TRP-1在黑素细胞及黑素瘤细胞的增殖及功能方面发挥重要作用,TRP-1反义核酸转染细胞细胞周期受到阻滞,酪氨酸酶活性明显降低。

关键词: 黑色素瘤, RNA,反义, 黑素细胞, 酪氨酸酶相关蛋白1

Abstract: Objective To construct the antisense eukaryotic vector of human TRP-1 (tyrosinase related protein 1) encoding gene, and transfect it into TRP-1 highly expressed melanocytes and malignant melanoma cell line, in order to further study the effects of antisense TRP-1 on the proliferation and functions of those cells. Methods TRP-1 cDNA was amplified by polymerase chain reaction (PCR) and the PCR products were subcloned into eukaryotic expression vector pcDNA3.1 on the opposite direction. Antisense recombinant vector was transfected into melanocytes and melanoma cell line. TRP-1 mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). TRP-1 protein level was detected by Western blot. Cell cycle was determined by flow cytometry. The activity of tyrosinase was valued by L-dopa reaction. Results The recombinant antisense vector pcDNA3.1/TRP-1(-) was constructed. Positive transfected cells could steadily express TRP-1 antisense RNA. It was showed that there was a low level of TRP-1 mRNA as indicated by RT-PCR, and a low level of TRP-1 protein as indicated by Western blot. Cell cycles were blocked in G1 stage. The suppress rates of tyrosinase was 46% in transfected melanocytes and 54% in malignant melanoma cells, respectively. Conclusions TRP-1 plays an important role in the proliferation and functions of melanocytes and melanoma cells. Antisense TRP-1 could block the cell cycles and decrease the activity of tyrosinese in those cells.

Key words: Melanoma, RNA, antisense, Melanocytes, Tyrosinase related protein 1