CAJ | 학술논문

目的研究低氧对人胚胎干细胞(hESC)生物学特性维持及印迹基因表达状态的影响.方法在低氧(5％O2)条件下持续培养FY-hES-7细胞并采用细胞免疫荧光染色、RT-PCR等方法对其胚胎干细胞特性进行鉴定.运用全基因组基因表达谱芯片技术及实时荧光定量PCR方法检测FY-hES-7早期(第32代)和晚期(第52代)细胞的55个印迹基因表达状态.所有检测同时以常氧(21％ O2)培养作为对照.结果低氧条件下长期培养的FY-hES-7细胞具有和常氧条件下培养相似的生物学特性,阳性表达干细胞表面标志SSEA-3、SSEA-4、TRA- 1-60、TRA-1-81及碱性磷酸酶(AKP),但细胞克隆形态较常氧培养下典型,且未分化状态保持时间较常氧条件下更长.低氧长期培养后FY-hES-7细胞的55个印迹基因中,有15个印迹基因(27.27％)表达发生了变化(上调8个,下调7个),而常氧培养后则有39个印迹基因表达水平有变化(70.91％)(上调21个,下调18个),氧浓度对印迹的影响差异具有统计学意义(P＜0.05).结论低氧更有利于hESC生物学特性的维持.长期培养能对hESC的印迹表达产生影响,但低氧培养条件下hESC的印迹稳定性要明显好于常氧培养.
목적 연구저양대인배태간세포(hESC)생물학특성유지급인적기인표체상태적영향.방법 재저양(5％O2)조건하지속배양FY-hES-7세포병채용세포면역형광염색、RT-PCR등방법대기배태간세포특성진행감정.운용전기인조기인표체보심편기술급실시형광정량PCR방법검측FY-hES-7조기(제32대)화만기(제52대)세포적55개인적기인표체상태.소유검측동시이상양(21％ O2)배양작위대조.결과 저양조건하장기배양적FY-hES-7세포구유화상양조건하배양상사적생물학특성,양성표체간세포표면표지SSEA-3、SSEA-4、TRA- 1-60、TRA-1-81급감성린산매(AKP),단세포극륭형태교상양배양하전형,차미분화상태보지시간교상양조건하경장.저양장기배양후FY-hES-7세포적55개인적기인중,유15개인적기인(27.27％)표체발생료변화(상조8개,하조7개),이상양배양후칙유39개인적기인표체수평유변화(70.91％)(상조21개,하조18개),양농도대인적적영향차이구유통계학의의(P＜0.05).결론 저양경유리우hESC생물학특성적유지.장기배양능대hESC적인적표체산생영향,단저양배양조건하hESC적인적은정성요명현호우상양배양.
Objective To investigate the effect of hypoxia on maintenance of biological characteristics and imprinted genes expression in human embryonic stem cells (hESCs).Methods The FY-hES-7 cells were cultured under hypoxic conditions( 5％ O2 ) followed by identification of features of stem cells by using cytologic fluorescent immunoassaying and reverse transcriptase polymerase chain reaction.Expression of 55 imprinted genes during the early ( 32nd passage) and late phase (52nd passage) was determined via whole genome expression genechip assaying and real-time polymerase chain reaction,with the cells cultured under normoxia condition (21％ O2) for control.Results Both FY-hES-7 cells cultured under hypoxia and normoxia conditions yielded comparable biological characteristics of hESCs and positive expression rates of stem cell surface markers,namely SSEA-3,SSEA-4,TRA-1-60,TRA-1-81 and alkaline phosphatase.Notably,the cells cultured under hypoxia condition were associated with typical cloning morphology and prolonged maintenance of undifferentiated state.Oxygen concentration was found as having more profound influence(P＜0.05) on 55 imprinted genes in FY-hES-7 cells cultured under normoxia condition,suggested by altered expression (21 up-regulated and 18 down-regulated) in 39 genes(70.91％),as compared with those cultured under hypoxia condition evidenced by varied expression(8 up-regulated and 7 down-regulated) in 15 genes(27.27％).Conclusion Hypoxia favors maintenance of the biological features of hESCs,and on the basis of prolonged culture,may result in marked impact on imprintedgene expression,as evidenced by higher stability when compared with those cultured under normoxia condition.