CAJ | 학술논문

目的观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法由胚胎大鼠脑组织获取NSCs并进行传代培养和分化鉴定,使用流式细胞术检测NSCs纯度及SDF-1特异性受体CXCR4的表达情况,之后利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0 ng/L、1 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、500 ng/mL和1000 ng/mL)对NSCs迁移数量的影响,并使用μ-载片观察SDF-1对NSCs迁移距离的影响.结果成功分离培养得到了能够在体外不断分裂增殖、具有多向分化潜能的NSCs,连续传3代后绝大部分细胞nestin表达阳性,nestin和CXCR4的共表达率达到80%左右.细胞趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500ng/mL时达到最高峰[(256.79±38.27)个细胞/每高倍视野].在μ-载片细胞生长通道两侧的SDF-1浓度梯度(500ng/mL→0ng/mL)作用下,由细胞克隆球迁出的细胞呈不对称分布,通常有更多的迁出细胞分布于趋化因子浓度较高的一侧,且该侧细胞的最大迁移距离也比对侧远.结论 SDF-1与其特异性受体CXCR4相巨作用能够诱导NSCs发生靶向性迁移.
목적 관찰기질세포연생인자-1(SDF-1)대신경간세포(NSCs)천이적영향.방법 유배태대서뇌조직획취NSCs병진행전대배양화분화감정,사용류식세포술검측NSCs순도급SDF-1특이성수체CXCR4적표체정황,지후이용Blind-Well소실체외천이체계관찰불동농도적SDF-1(0 ng/L、1 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、500 ng/mL화1000 ng/mL)대NSCs천이수량적영향,병사용μ-재편관찰SDF-1대NSCs천이거리적영향.결과성공분리배양득도료능구재체외불단분렬증식、구유다향분화잠능적NSCs,련속전3대후절대부분세포nestin표체양성,nestin화CXCR4적공표체솔체도80%좌우.세포추화실험결과표명,SDF-1대NSCs유교강적추화작용,수착SDF-1농도적승고,발생천이적세포수량야수지증가,병우SDF-1농도위500ng/mL시체도최고봉[(256.79±38.27)개세포/매고배시야].재μ-재편세포생장통도량측적SDF-1농도제도(500ng/mL→0ng/mL)작용하,유세포극륭구천출적세포정불대칭분포,통상유경다적천출세포분포우추화인자농도교고적일측,차해측세포적최대천이거리야비대측원.결론 SDF-1여기특이성수체CXCR4상거작용능구유도NSCs발생파향성천이.
Objective To investigate the effect ofstromal cell derived factor-1 (SDF-1) on the regulation of neural stem cells (NSCs) migration.Methods NSCs were obtained from the cerebral cortex of embryonic rats and cultured in serum-free medium,and their stem cell properties were assessed by means of induced differentiation in vitro into neurons and astrocytes.After in vitro cell culture,the purity of NSCs and the co-expression rate of CXCR4/nestin were detected by flow cytometry.Blind-well chambers were employed to detect the chemotactic effects of SDF-1 by counting the cells which had crossed a 8 μm pore membrane when confronted with varying concentrations of SDF-1 (0,1,10,50,100,500 and 1000 ng/mL),and the distribution of cells migrated out of the same neurosphere was overviewed by μ-slides in the persistent concentration gradient of SDF-1.Results Neurospheres were formed by persistent proliferation of NSCs, which were capable of differentiating into neurons (β-tubulin+) and astrocytes (GFAP+) in media without mitogens,and flow cytometry analyses showed that most of the cultured cells expressed nestin and the co-expression rate of CXCR4/nestin was nearly 80%.SDF-1 showed great chemotaxis to NSCs,and the amount of cells having migrated through the membrane in 500 ng/ml SDF-1 group was higher than that in other groups (P<0.05).When the cells were confronted with a linear concentration gradient (from 500 to 0 ng/mL),which was generated by diffusion and stable for at least 48 h,the cells migrated out ofa neruosphere could distribute irregularly with more cells locating in the region of higher concentration of SDF-1 and longer migration distance away from the center of the neurosphere than the opposite.Conclusion SDF-1 binding to its specific receptor CXCR4 was capable of inducing NSCs migrating directionally to the source of SDF-1.