CAJ | 학술논문

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。
목적：검측여간세포상관적2충인자배태간세포상관전록인자4（Nanog）화분화항원족44（CD44）재위암세포주MKN45현부구체세포중적표체。방법선택인위암세포주MKN45，재함유표피생장인자（EGF）급성섬유생장인자（bFGF）무혈청배양액중배양，관찰구체형성정황；채용면역인적（Western blot）、면역형광여실시정량취합매련반응（qRT-PCR）검측간세포상관기인Nanog화CD44재구체세포급원대첩벽세포중적표체차이。결과위암세포주MKN45재무혈청배양조건하능형성현부구체，구체세포중적Nanog화CD44적mRNA상대표체량분별위2.34±0.22화1.18±0.04，명현고우첩벽세포중적Nanog화CD44적mRNA상대표체량1.00±0.00화1.00±0.05；구체세포중적Nanog화CD44적단백질상대표체량분별위0.18±0.02화0.24±0.04，명현고우첩벽세포중적상대표체량0.07±0.02화0.18±0.01（P＜0.05）。구체세포중Nanog주요분포재세포핵주위급세포질중，이CD44주요재세포막상표체，차동일개구체세포중적Nanog여CD44대다동시표체。결론위암세포주MKN45재무혈청배양조건하형성적구체세포구유종류간세포적특성。Nanog/CD44공표체세포가능시위암간세포적일충표형。
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.