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纸质出版:2014
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NANOG基因在侧群表型干细胞样肝癌细胞中的表达及意义[J]. 中山大学学报(医学科学版), 2014,35(1).
Significance of NANOG Gene Expression in SP Phenotype Stem Cell-like Hepatoma Cells[J]. Journal of Sun Yat-sen University (Medical Sciences), 2014, 35(1).
摘 要: 【目的】 基于侧群(SP)细胞分选方法,从肝癌细胞Huh7和Hep3B中分选SP表型干细胞样肝癌细胞,观察干细胞标记物NANOG基因在该SP表型肝癌细胞中的表达。【方法】 采用流式细胞术检测和分选肝癌细胞Huh7和Hep3B中的SP细胞和非侧群(NSP)细胞,细胞生长曲线法检测SP和NSP细胞的增殖能力,Transwell侵袭实验检测SP和NSP细胞的侵袭能力,MTT法检测SP和NSP细胞对5-氟尿嘧啶(5-FU)和阿霉素(DOX)的药物敏感性;Real-time PCR和WB检测SP与NSP细胞中NANOG基因的表达。【结果】肝癌细胞Huh7和Hep3B中分选的SP细胞比例分别为(5.3 ± 0.8)%和(13.49 ± 1.0)%。生长曲线表明Huh7和Hep3B的SP细胞的增殖速度快于NSP细胞(P < 0.05),体外侵袭实验结果显示Huh7和Hep3B的SP细胞体外侵袭能力高于NSP细胞(P < 0.05),MTT结果显示Huh7和Hep3B SP细胞对5-FU和DOX有耐药性,高于NSP细胞(P < 0.05)。Huh7和Hep3B SP细胞NANOG mRNA平均表达水平分别是NSP细胞的4.17和5.51倍(P < 0.05),NANOG蛋白平均表达水平分别是NSP细胞的3.78和5.01倍(P < 0.05)。【结论】 肝癌细胞Huh7和Hep3B中分选的SP细胞符合肿瘤干细胞的部分生物学特征,NANOG基因在SP表型干细胞样肝癌细胞中高表达。
Abstract: 【Objective】 To isolate and identify the biological characteristics of stem celllike hepatoma cells from the hepatoma cell line Huh7 and Hep3B based on side population (SP) cell sorting method, and to study the expression of NANOG gene in the SP phenotype stem celllike hepatoma cells. 【Methods】 Flow cytometry was used to sort SP cells and nonside population (NSP) cells form hepatoma cell line Huh7 and Hep3B, the proliferation ability of SP cells and NSP cells were detected by cell growth curve, the invasive properties of SP cells and NSP cells were tested by transwell assay, the chemotherapeutic drug sensitivity of SP cells and NSP cells towards 5fluorouracil and doxorubicin were tested by MTT assay, the expression levels of NANOG mRNA and protein in SP cells and NSP cells were detected by realtime PCR and Western blot analysis. 【Results】 (5.3 ± 0.8)% and (13.49 ± 1.0)% SP cells were sorted from Hepatoma cell line Huh7 and Hep3B, respectively. Growth curve assay showed that the proliferation ability of Huh7 and Hep3B SP cells was higher than that of NSP cells. Transwell assay showed that the invitro invasion capacity of Huh7 and Hep3B Spcells was significantly higher than that of NSP cells (P < 0.05). MTT assay showed that Huh7 and Hep3B SP cells have stronger drug tolerance to 5fluorouracil and doxorubicin than NSP cells (P < 0.05). The expression levels of NANOG mRNA in Huh7 andHep3B SP cells were 4.17 and 5.51 times of that in NSP cells, respectively (P < 0.05). The expression levels of NANOG protein in Huh7 and Hep3B SP cells were 3.78 and 5.01 times of that in NSP cells, respectively (P < 0.05). 【Conclusions】 The SP cells sorted form Hepatoma cell line Huh7 and Hep3B have some of the characteristics of cancer stem cells, and NANOG gene is highly expressed in these SP phenotype stem celllike hepatoma cells.
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