生理学报Acta Physiologica Sinica, June 25, 2003, 55(3)

研究论文

人体肝癌细胞急性低氧及低氧习服差异表达基因分析

王金惠*, 善亚君, 从玉文, 吴岚军, 苑晓玲, 赵振虎, 王升启, 陈家佩

军事医学科学院放射医学研究所, 北京 100850

摘要: 本文分析了人体肝癌细胞(HepG2)急性低氧处理以及低氧习服处理后基因表达谱的改变。急性低氧处理为细胞在1%氧气中培养48 h, 低氧习服处理为细胞在1%氧气中培养24 h, 常氧培养24 h, 以此作为一个周期, 重复6个周期。联合应用抑制消减杂交技术和cDNA芯片技术, 筛选HepG2细胞经急性低氧处理与正常培养细胞相比差异表达的基因, 以及经低氧习服处理细胞与正常培养细胞相比差异表达的基因。 结果显示, HepG2细胞经急性低氧处理与在常氧条件下培养相比, 差异表达的基因有37个, 表达水平全部表现为下调, 其中包括参与细胞周期、 细胞应激、 细胞信号转导、 细胞骨架形成、 转录相关蛋白及细胞代谢相关蛋白的基因, 1个未知基因序列、 4个EST序列、 5个线粒体蛋白基因, 另外有功能不明的蛋白质基因12个。低氧习服处理的细胞与常氧条件下培养的细胞相比, 差异表达的基因有6个, 其中包括两个线粒体蛋白基因、 金属蛋白酶1基因、 转铁蛋白基因、 Thymosin.beta-4和TPT1基因。其中线粒体蛋白ND4、 转铁蛋白、 Thymosin.beta-4和TPT1基因的表达呈上调, 线粒体ND1及金属蛋白酶1基因的表达水平呈下调。经低氧习服处理后, 细胞低氧耐受力提高, 低氧习服处理细胞基因的表达与急性低氧处理细胞和正常培养细胞的基因表达不同, 这种变化可能与低氧习服细胞低氧耐受力的增强有关。

关键词: 细胞低氧; 抑制消减杂交; cDNA微阵列; 基因表达

中图分类号: Q493.9

Identification of differentially expressed genes of acute hypoxia-treated HepG2 cells and hypoxia-acclimatized HepG2 cells

WANG Jin-Hui*, SHAN Ya-Jun, CONG Yu-Wen, WU Lan-Jun, YUAN Xiao-Ling, ZHAO Zhen-Hu, WANG Sheng-Qi, CHEN Jia-Pei

Institute of Radiation Medicine Sciences, Academy of Military Medical Sciences, Beijing 100850

Abstract: To provide necessary information for further understanding of molecular mechanism of hypoxia acclimatization, the differentially expressed genes of HepG2 cells exposed to normoxia, acute hypoxia-treated cells which were exposed to 1% oxygen for 48 h, and hypoxia-acclimatized HepG2 cells which were cultured for 6 circles of alternate low oxygen (1% oxygen for 24 h) and normal oxygen (21% oxygen for 24 h), were identified respectively by combining the suppression subtractive hybridization (SSH) and cDNA microarray. Thirty-seven genes were expressed differentially in cells exposed to 1% oxygen for 48 h compared with those in cells exposed to normoxia. The expression of all these 37 genes was down-regulated, including the genes participating in cell cycle, cell response to stimulus, and cell signal transduction, and cell cytoskeleton formation, the genes associated with transcription and cell metabolism, 4 expressed sequence tags (ESTs), and 12 genes of which the functions are not known. There is a novel gene sequence, which has not been found in existing databases. There were only 6 genes differentially expressed in the hypoxia-acclimatized cells compared with cells exposed to normoxia, including two mitochondrion genes, metalloprotease-1 gene, ferritin gene, thymosin beta-4 and TPT1 genes. The expressions of mitochondrion ND4, ferritin, thymosin beta-4 and TPT1 were up-regulated, while expressions of mitochondrion ND1 gene and metalloproease-1 gene were down-regulated. Cell tolerance to hypoxia increased after the cells were hypoxia-acclimatized. The different gene expression patterns of the acute hypoxia-treated cells and the hypoxia-acclimatized cells may be related to the increased tolerance of the cells to hypoxia.

Key words: cell hypoxia; suppression subtractive hybridization; cDNA microarray; gene expression