中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (6): 386-389.

• 论著 • 上一篇    下一篇

NHERF1及β联蛋白在黑素瘤细胞系A375、M14、MV3中的表达及意义

张韡1,程芳1,2,张磊2,吴侃3,黄莹雪4,周武庆3,3,姜祎群曾学思5,孙建方5   

  1. 1. 中国医学科学院皮肤病研究所
    2.
    3. 中国医学科学院北京协和医学院皮肤病研究所
    4. 中南大学湘雅医院皮肤科
    5. 南京 中国医学科学院北京协和医学院皮肤病研究所
  • 收稿日期:2012-12-04 修回日期:2013-02-24 出版日期:2013-06-15 发布日期:2013-06-01
  • 通讯作者: 孙建方 E-mail:fangmin5758@aliyun.com
  • 基金资助:
    2011年协和青年基金

Expressions of NHERF1 and β-catenin in malignant melanoma cell lines （A375, M14 and MV3） and their significance

  • Received:2012-12-04 Revised:2013-02-24 Online:2013-06-15 Published:2013-06-01

PDF

10

赞

摘要： 【摘要】 目的 探讨钠氢交换子调节因子-1（NHERF1）及β联蛋白在黑素瘤细胞系A375、M14、MV3中的表达及意义。方法 使用免疫细胞化学法、实时荧光定量-PCR法、免疫印迹法对黑素瘤细胞系A375、M14、MV3及黑素细胞中NHERF1及β联蛋白的表达情况进行观察。使用MTT法对黑素瘤细胞系A375、M14、MV3及黑素细胞的增殖情况进行检测。结果 NHERF1及β联蛋白在黑素细胞中同时表现为细胞膜阳性表达，而在黑素瘤细胞系A375、M14、MV3中均出现膜表达的缺失，同时呈现不同程度的胞质和（或）胞核阳性表达。在3株黑素瘤细胞系中，MV3细胞中的NHERF1免疫细胞化学染色仅呈少部分胞质弱阳性表达，实时荧光定量PCR检测NHERF1的mRNA表达水平最低，免疫印迹法中未见NHERF1蛋白表达条带，而其在MTT法中检测到的增殖速度最快，明显高于另外两株细胞。A375细胞中的NHERF1免疫细胞化学染色呈胞质及胞核的强阳性表达，实时荧光定量PCR检测NHERF1的mRNA表达水平最高，免疫印迹法中可见NHERF1蛋白最强阳性表达，而其增殖速度却慢于MV3细胞。β联蛋白在MV3细胞中免疫细胞化学染色呈细胞质及细胞核的强阳性表达，实时荧光定量PCR检测的mRNA表达水平最高，免疫印迹法检测到蛋白最强阳性表达；而A375细胞及M14细胞中，β联蛋白在免疫细胞化学染色呈相对弱的细胞质阳性表达，实时荧光定量PCR检测的mRNA表达水平明显较低；免疫印迹法中的蛋白表达较MV3中的表达低。结论 黑素瘤细胞胞质中NHERF1表达量可能与细胞的增殖速度相关，NHERF1在黑素瘤细胞中表达程度越低细胞增殖越快。黑素瘤细胞中的NHERF1与β联蛋白存在着差异性表达。 【关键词】 黑色素瘤； 钠氢交换子调节因子-1； β连环蛋白

关键词: 黑色素瘤, 钠氢交换子调节因子-1, β-连环蛋白

Abstract: ZHANG Wei, CHENG Fang, ZHANG Lei, WU Kan, HUANG Ying-xue, ZHOU Wu-qing, JIANG Yi-qun, ZENG Xue-si, SUN Jian-fang. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College，Nanjing 210042, China Corresponding author: SUN Jian-fang, Email: fangmin5758@yahoo.com.cn 【Abstract】 Objective To investigate the expressions of Na+/H+ exchanger regulatory factor 1 （NHERF1） and β-catenin in human melanoma cell lines A375, M14 and MV3, as well as their significance. Methods Three human melanoma cell lines A375, M14, MV3 as well as melanocytes isolated from a healthy 8-year-old boy were cultured in vitro. Real-time fluorescence-based quantitative PCR was performed to detect the mRNA expressions of NHERF1 and β-catenin, immunocytochemistry and Western blot to quantify the protein expression of NHERF1 and β-catenin in these cells, and methyl thiazolyl tetrazolium （MTT） assay to evaluate the proliferative activity of these cells. Results NHERF1 and β-catenin were expressed in the membrane of melanocytes, and in the cytoplasm and/or nuclei, but not in the membrane of these melanoma cell lines. Of the three melanoma cell lines, only a small part of MV3 cells showed a weak immunocytochemical expression of NHERF1 in cytoplasm with the lowest expression level of NHERF1 mRNA, whereas A375 cells exhibited the strongest immunocytochemical expression of NHERF1 in cytoplasm and nuclei with the highest expression level of NHERF1 mRNA. As Western blot analysis showed, NHERF1 protein was undetectable in MV3 cells, but strongly expressed in A375 cells. The proliferation of MV3 cells was faster than that of A375 cells and M14 cells. Meanwhile, MV3 cells displayed the strongest immunocytochemical expression of β-catenin in cytoplasm and nuclei, with the highest level of mRNA and protein expression of β-catenin; A375 and M14 cells showed a relatively weaker expression of cytoplasmic β-catenin, with a decrease in the expression intensity of β-catenin mRNA and protein. Conclusions The cytoplasmic expression level of NHERF1 may be associated with the proliferative activity of melanoma cells, and it seems that the lower the expression level of NHERF1, the faster the cell proliferation. NHERF1 and β-catenin are differentially expressed in these melanoma cell lines. 【Key words】 Melanoma; NHERF1; β-catenin