天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (1): 30-33.doi: 10.3969/j.issn.0253-9896.2015.01.008

• 细胞与分子生物学 • 上一篇    下一篇

Nanog 与 CD44 在胃癌细胞株 MKN45 球体细胞中的表达及其意义

刘建明， 周友浪△， 马利林， 徐骏飞， 章建国#br#   

  1. 南通大学附属医院普外科（邮编 226001）
  • 收稿日期:2014-04-21 修回日期:2014-08-20 出版日期:2015-01-15 发布日期:2015-01-30
  • 通讯作者: 周友浪△通讯作者 E-mail： liujmtq1@163.com E-mail:mall90522@163.com
  • 基金资助:
    江苏省南通市科技局资助项目（HS2012067）

The expression and significance of Nanog and CD44 in spheroid body-forming cells of gastric cancer cell line MKN-45

LIU Jianming, ZHOU Youlang△, MA Lilin, XU Junfei, ZHANG Jianguo#br# #br#   

  1. Department of General Surgery, Affiliated Hospital of Nantong University, Jiangsu 226001, China
  • Received:2014-04-21 Revised:2014-08-20 Published:2015-01-15 Online:2015-01-30
  • Contact: ZHOU Youlang △Corresponding Author E-mail:liujmtq1@163.com E-mail:mall90522@163.com

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摘要： 摘要： 目的 检测与干细胞相关的 2 种因子胚胎干细胞相关转录因子 4（Nanog）和分化抗原簇 44（CD44）在胃癌细胞株 MKN45 悬浮球体细胞中的表达。 方法 选择人胃癌细胞株 MKN45， 在含有表皮生长因子（EGF）及成纤维生长因子（bFGF）无血清培养液中培养， 观察球体形成情况； 采用免疫印迹（Western blot）、免疫荧光与实时定量聚合酶链反应（qRT-PCR）检测干细胞相关基因 Nanog 和 CD44 在球体细胞及原代贴壁细胞中的表达差异。 结果 胃癌细胞株 MKN45 在无血清培养条件下能形成悬浮球体， 球体细胞中的 Nanog 和 CD44 的 mRNA 相对表达量分别为 2.34±0.22 和 1.18±0.04， 明显高于贴壁细胞中的 Nanog 和 CD44 的 mRNA 相对表达量 1.00±0.00 和 1.00±0.05；球体细胞中的 Nanog 和 CD44 的蛋白质相对表达量分别为 0.18±0.02 和 0.24±0.04， 明显高于贴壁细胞中的相对表达量 0.07±0.02 和 0.18±0.01（P＜ 0.05）。 球体细胞中 Nanog 主要分布在细胞核周围及细胞质中， 而 CD44 主要在细胞膜上表达， 且同一个球体细胞中的 Nanog 与 CD44 大多同时表达。 结论 胃癌细胞株 MKN45 在无血清培养条件下形成的球体细胞具有肿瘤干细胞的特性。 Nanog/CD44 共表达细胞可能是胃癌干细胞的一种表型。

关键词: 胃肿瘤, 肿瘤干细胞, 抗原, CD44, 悬浮培养法, Nanog

Abstract: Abstract： Objective To detect the expression of stem-cell related factors Nanog and CD44 in spheroid body-form⁃ ing cells of gastric cancer cell line MKN-45. Methods The gastric cancer cell line MKN-45 was used to culture spheroid bodies in non-adherent condition in a serum-free medium supplemented with epidermal growth factor (EGF) and basic fibro⁃ blast growth factor (bFGF). Using Western blot analysis, immunofluorescence staining and quantitative real time polymerase chain reaction（qRT-PCR）, the expression levels of stem cell-related genes Nanog and CD44 were studied. Results In this study, we observed that MKN45 cells formed spheroid bodies in non-adherent condition in a serum-free medium, and the levels of Nanog and CD44 mRNA expression in spheroid body-forming cells were 2.34±0.22 and 1.18±0.04， respectively, which were higher than those in parental cells (1.00±0.00 and 1.00±0.05). The levels of Nanog and CD44 protein expression in spheroid body-forming cells were 0.18±0.02 and 0.24±0.04, respectively, which were significantly higher than those in pa⁃ rental cells (0.07±0.02 and 0.18±0.01, P＜ 0.05). Nanog protein was positively stained within the perinuclear and cytoplasm of the spheroid body-forming cells, and CD44 was positively stained mainly in the membrane. Dual staining of Nanog/CD44 indicated that the embryonal protein Nanog was co-localized with CD44 in the spheroid body-forming cells. Conclusion Spheroid body-forming cells developed from human gastric cancer cell line MKN-45 in serum-free medium supplemented with EGF and bFGF show characteristics of cancer stem cell (CSC). The cells co-expressed of CD44 and Nanog maybe a phe⁃ notype of gastric CSCs.

Key words: stomach neoplasms, neoplastic stem cells, antigens, CD44, suspension culture, Nanog