CAJ | 학술논문

目的：整体比较2种促进诱导性多能干细胞（ induced pluripotent stem cells ， iPSC）向神经干细胞（neural stem cells，NSC）分化的方法，确定一种稳定、高效的获得NSC的方法，并对NSC进行系统鉴定。方法：方法A：SB431542和drosomophorin的浓度均为5μmmol／L，诱导初始密度100％；方法B：SB431542的浓度为5 mmol／L， drosomophorin的浓度为1 mmol／L，诱导初始密度为40％。比较及鉴定方法：镜下观察诱导获得NSC的状态；real-time PCR比较神经干细胞相关基因Pax6、nestin、Sox1、Sox2等表达量；流式细胞术分析诱导第16天Pax6阳性率；免疫荧光定性分析神经干细胞相关蛋白的表达及其自发分化的能力。结果：方法A获得的NSC悬起后成球趋势明显，圆形，透明；方法B诱导获得NSC形状不规则，色灰暗。 Real-time PCR结果证明方法A诱导获得的细胞神经干细胞相关基因的表达量高于方法B。流式细胞术分析证明第16天，PAX6的阳性率，方法A高于方法B。经鉴定，方法A获得的神经干细胞高表达Pax6、nestin、Sox2等基因自发分化30 d，形成明显的神经纤维束，表达TUJ-1、MAP2及GFAP等神经元和胶质细胞的特异性标志物。结论：方法A整体优于方法B，我们推荐方法A作为诱导iPSC向神经干细胞分化的方法。
목적：정체비교2충촉진유도성다능간세포（ induced pluripotent stem cells ， iPSC）향신경간세포（neural stem cells，NSC）분화적방법，학정일충은정、고효적획득NSC적방법，병대NSC진행계통감정。방법：방법A：SB431542화drosomophorin적농도균위5μmmol／L，유도초시밀도100％；방법B：SB431542적농도위5 mmol／L， drosomophorin적농도위1 mmol／L，유도초시밀도위40％。비교급감정방법：경하관찰유도획득NSC적상태；real-time PCR비교신경간세포상관기인Pax6、nestin、Sox1、Sox2등표체량；류식세포술분석유도제16천Pax6양성솔；면역형광정성분석신경간세포상관단백적표체급기자발분화적능력。결과：방법A획득적NSC현기후성구추세명현，원형，투명；방법B유도획득NSC형상불규칙，색회암。 Real-time PCR결과증명방법A유도획득적세포신경간세포상관기인적표체량고우방법B。류식세포술분석증명제16천，PAX6적양성솔，방법A고우방법B。경감정，방법A획득적신경간세포고표체Pax6、nestin、Sox2등기인자발분화30 d，형성명현적신경섬유속，표체TUJ-1、MAP2급GFAP등신경원화효질세포적특이성표지물。결론：방법A정체우우방법B，아문추천방법A작위유도iPSC향신경간세포분화적방법。
AIM:To select an efficient way of promoting induced pluripotent stem cells ( iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods.METHODS:The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100%initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density.For comparison and identification of the 2 methods, the growth state was observed under microscope , and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry .The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis .RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax 6, nestin, Sox1 and Sox2.The growth state was better and the cells differentiated into neurons and astrocytes normally .CONCLU-SION:The method A was superior to method B , and we recommend the method A with 5 mmol/L of SB431542 and droso-mophorin and 100%initial cell density as the method for differentiating NSC .