CAJ | 학술논문

目的研究p27~(Kip1)在永生化人神经前体细胞分化中的作用,探讨神经前体细胞的分化机制.方法取本课题组已建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)进行培养,在细胞进入对数生长期时给予1μmol/L全反式视黄酸(RA)诱导,重复诱导3次,每次均在诱导的第3、5、7天收集细胞,用流式细胞仪分析RA诱导第3天细胞周期的变化,用免疫印迹法检测RA诱导第3、5、7天p27~(Kip1)、p21~(cip1)细胞周期蛋白依赖激酶2(cdk2)及S期激酶相关蛋白2(skp2)的变化.结果流式细胞术结果显示,hSN12W-TERT细胞经1μmol/L RA诱导3d后,G0/G1期细胞的比例由77.25%上升至85.68%,而S期的比例由9.38%下降到8.57%.免疫印迹法结果显示,RA诱导第3天,hSN12W-TERT细胞p27~(Kip1)蛋白表达比未经RA诱导的细胞增加,并在RA诱导第5天达到高峰(P<0.05).未经RA诱导的正常hSN12W-TERT细胞p21~(cip1)蛋白表达弱,RA诱导后p21~(cip1)蛋白的表达略呈下降趋势.RA诱导前后hSN12W-TERT细胞cdk2蛋白的表达变化不明显,但反映cdk2活性的磷酸化cdk2(p-cdk2)的表达在RA诱导后明显下降,同时,参与p27~(Kip1)泛素降解途径的重要因子skp2的表达在RA诱导后明显下降.结论在RA诱导hSN12W-TERT细胞分化的过程中,p27~(Kip1)通过抑制cdk2的活性而发挥促细胞分化的作用,且RA诱导后p27~(Kip1)蛋白含量增加与其泛素降解途径被抑制密切相关.
목적 연구p27~(Kip1)재영생화인신경전체세포분화중적작용,탐토신경전체세포적분화궤제.방법 취본과제조이건립적영생화인신경전체세포계hSN12W-TERT세포(제12대)진행배양,재세포진입대수생장기시급여1μmol/L전반식시황산(RA)유도,중복유도3차,매차균재유도적제3、5、7천수집세포,용류식세포의분석RA유도제3천세포주기적변화,용면역인적법검측RA유도제3、5、7천p27~(Kip1)、p21~(cip1)세포주기단백의뢰격매2(cdk2)급S기격매상관단백2(skp2)적변화.결과 류식세포술결과현시,hSN12W-TERT세포경1μmol/L RA유도3d후,G0/G1기세포적비례유77.25%상승지85.68%,이S기적비례유9.38%하강도8.57%.면역인적법결과현시,RA유도제3천,hSN12W-TERT세포p27~(Kip1)단백표체비미경RA유도적세포증가,병재RA유도제5천체도고봉(P<0.05).미경RA유도적정상hSN12W-TERT세포p21~(cip1)단백표체약,RA유도후p21~(cip1)단백적표체략정하강추세.RA유도전후hSN12W-TERT세포cdk2단백적표체변화불명현,단반영cdk2활성적린산화cdk2(p-cdk2)적표체재RA유도후명현하강,동시,삼여p27~(Kip1)범소강해도경적중요인자skp2적표체재RA유도후명현하강.결론 재RA유도hSN12W-TERT세포분화적과정중,p27~(Kip1)통과억제cdk2적활성이발휘촉세포분화적작용,차RA유도후p27~(Kip1)단백함량증가여기범소강해도경피억제밀절상관.
Objective To investigate whether there is any functional link between p27~(Kip1) function and all-trans retinoic acid (RA) in the control of neuronal differentiation of immortalized human neural progenitor cells (hSN12W-TERT cells). To investigate the mechanism by which p27~(Kip1) regulates the differentiation of immortalized human neural progenitor cells. Methods hSN12W-TERT cells were derived from the striatums of human embryos at 12 weeks gestation and cultured with serum-free medium in presence of EGF and bFGF. At the appropriated time, hSN12W-TERT cells were exposed to 1μmol/L RA for 3, 5, 7 days respectively. The experiment was repeated there times. Cell cycle analysis was performed by flow cytometry analysis (FACS). The expression of p27~(Kip1), p21~(cip1), cyclin-dependent kinase 2 (cdk2), p-cdk2 and S-phase kinase-associated protein 2 (skp2) in hSN12W-TERT cells before and after RA treatment cells were determined by using Western blotting analysis. Results FACS result showed that 77.25% of proliferating hSN12W-TERT cells were in the G1/G0-phase while 9.38% of cells in the S-phase. Following RA treatment, cell growth was arrested, and 85.68% of cells accumulated in G1/G0-phase while 8.57% of cells in the S-phase. Western blotting analysis demonstrated that the levels of p27~(Kip1) in the hSN12W-TERT cells increased following 3 days' treatment with RA compared with those of normal untreated cells, with a peak at 5 days (P<0.05). The similar results were acquired both in nuclear proteins and in cytoplasm proteins of hSN12W-TERT cells. The expression level of p21~(cip1) decreased in response to RA treatment. RA did not affect the expression of cdk2, but the expression of p-cdk2, which represented the activity of cdk2, was markedly decreased in response to RA treatment. Skp2, which was required for the ubiquitin-mediated degradation of p27~(Kip1), was detected in proliferating hSN12W-TERT cells. The expression of skp2 reduced dramatically in response to RA treatment in a time-dependent manner.Conclusion There is a functional link between RA and p27~(Kip1) function in the control of neuronal differentiation in hSN12W-TERT cells. P27~(Kip1) plays a key role during neuronal differentiation. Moreover, high levels of p27~(Kip1) are associated with its degradation inhibiting through reducing proteasome-dependent proteolysis.