CAJ | 학술논문

目的观察全反式维甲酸(ATRA)对胶质瘤干细胞表型的影响.方法首先神经球培养法富集培养原代胶质瘤干细胞(GSC)；以ATRA分化培养1周；用流式细胞术对比检测GSC与其分化细胞CD133阳性比例变化；免疫印迹法对比分析干细胞标志物及分化标志物表达水平差异；逆转录聚合酶链式反应检测Notch1活性变化；基于Heochst33342外排法流式侧群细胞比例分析和流式细胞周期检测分析；最后将GSC与其分化细胞分别原位种植鼠脑,对比观察裸鼠存活时间.结果神经球培养法有效富集培养原代胶质瘤干细胞球,经ATRA诱导分化1周,细胞呈多突起伸展样贴壁生长；流式细胞术检测示ATRA分化处理降低CD133阳性细胞比例(由平均33.55％降至13.49％,P＜0.05)；免疫印迹检测示ATRA分化处理降低CD133和Nestin表达水平,而标志物GFAP表达增加(P ＜0.05)；RT-PCR检测示Notch1经ATRA分化后表达明显降低(P＜0.05)；流式侧群分析示ATRA处理后侧群细胞比例显著降低(由平均0.9％降至0.3％)；经ATRA处理后,细胞周期阻滞于G0/G1期；ATAR诱导分化后裸鼠生存时间明显延长(P＜0.05).结论 ATRA可有效分化GSC,降低GSC干细胞表型,延长裸鼠生存时间,提示ATRA诱导GSC分化在胶质瘤治疗中的可能应用前景.
목적 관찰전반식유갑산(ATRA)대효질류간세포표형적영향.방법 수선신경구배양법부집배양원대효질류간세포(GSC)；이ATRA분화배양1주；용류식세포술대비검측GSC여기분화세포CD133양성비례변화；면역인적법대비분석간세포표지물급분화표지물표체수평차이；역전록취합매련식반응검측Notch1활성변화；기우Heochst33342외배법류식측군세포비례분석화류식세포주기검측분석；최후장GSC여기분화세포분별원위충식서뇌,대비관찰라서존활시간.결과 신경구배양법유효부집배양원대효질류간세포구,경ATRA유도분화1주,세포정다돌기신전양첩벽생장；류식세포술검측시ATRA분화처리강저CD133양성세포비례(유평균33.55％강지13.49％,P＜0.05)；면역인적검측시ATRA분화처리강저CD133화Nestin표체수평,이표지물GFAP표체증가(P ＜0.05)；RT-PCR검측시Notch1경ATRA분화후표체명현강저(P＜0.05)；류식측군분석시ATRA처리후측군세포비례현저강저(유평균0.9％강지0.3％)；경ATRA처리후,세포주기조체우G0/G1기；ATAR유도분화후라서생존시간명현연장(P＜0.05).결론 ATRA가유효분화GSC,강저GSC간세포표형,연장라서생존시간,제시ATRA유도GSC분화재효질류치료중적가능응용전경.
Objective To explore the effects of all-trans retinoic acid (ATRA) on glioma stem cell phenotype.Methods The glioma stem cell (GSC) from surgically resected human glioma specimens were isolated and enriched by neurosphere assay and then its differentiation was induced with all-trans retinoic acid (ATRA,1 μmol/L) for 1 week.Markers were determined by flow cytometry,Western blot and reverse transcription-polymerase chain reaction (RT-PCR).Side population cells were analyzed by flow cytometry.Growth characteristics were detected by neurosphere formation assay and cell cycle analysis.GSC and the differentiated cells (1 × 105) were implanted stereotactically and intracranially into the Balb/c nude mice to compare the survival time.All data were analyzed with the SPSS software version 17.0.Results ATRA potently induced the differentiation of GSC and reduced glioma stem cell phenotype.And there were an elevated expression of glial fibrillary acidic protein (GFAP) and a reduced expression of such stem cell makers as CD133 and Nestin.The side population rate decreased.ATRA inhibited the neurosphere formation of GSC and induced the arrest of cell growth.ATRA could prolong the survival time.Conclusion GSC may be differentiated efficiently by ATRA.The stemness of GSC decreases obviously after the differentiation of ATRA and the survival time is prolonged.Thus ATRA may be applied for targeted therapies of glioma stem cell.