CAJ | 학술논문

目的观察神经干细胞( NSC)向胶质瘤干细胞(GSC)及其分化细胞的迁移能力,探讨其趋化机制。方法干细胞条件培养U251和3例原代胶质瘤干细胞,以流式细胞术和Western blot对其鉴定;Transwell小室法检测GSC和其分化细胞条件培养液(CM)对NSC迁移的趋化能力;酶联免疫吸附试验(ELISA)法检测CM中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的分泌水平,并以表皮生长因子(EGF)、bFGF为对照分析CM对NSC的化学趋化作用;进一步以Dio和Dil分别标记NSC和GSC,体外混合培养观察NSC向GSC的迁移、以及对肿瘤干细胞球生长的影响。结果干细胞培养条件下的胶质瘤干细胞球,高表达干细胞标志物Nestin和/或CD133( 11.02％～ 33.55％);分化后干细胞标志物表达下降,分化标志物胶质纤维酸性蛋白(GFAP)表达增加(P＜0.05);GSC与其分化细胞比较,分泌高水平的趋化因子VEGF和bFGF(P ＜0.05),并对NSC有高度趋化能力;体外混合接触培养也显示NSC向肿瘤干细胞球的迁移和包绕,并且能够显著抑制肿瘤干细胞球的生长(P＜0.05)。结论 GSC体外可趋化NSC向其迁移,其趋化作用较分化的肿瘤细胞更为显著,并且与其分泌高水平的生长因子有关;向肿瘤干细胞球迁移的NSC可抑制其体外生长。
목적 관찰신경간세포( NSC)향효질류간세포(GSC)급기분화세포적천이능력,탐토기추화궤제。방법 간세포조건배양U251화3례원대효질류간세포,이류식세포술화Western blot대기감정;Transwell소실법검측GSC화기분화세포조건배양액(CM)대NSC천이적추화능력;매련면역흡부시험(ELISA)법검측CM중혈관내피생장인자(VEGF)、감성성섬유세포생장인자(bFGF)적분비수평,병이표피생장인자(EGF)、bFGF위대조분석CM대NSC적화학추화작용;진일보이Dio화Dil분별표기NSC화GSC,체외혼합배양관찰NSC향GSC적천이、이급대종류간세포구생장적영향。결과 간세포배양조건하적효질류간세포구,고표체간세포표지물Nestin화/혹CD133( 11.02％～ 33.55％);분화후간세포표지물표체하강,분화표지물효질섬유산성단백(GFAP)표체증가(P＜0.05);GSC여기분화세포비교,분비고수평적추화인자VEGF화bFGF(P ＜0.05),병대NSC유고도추화능력;체외혼합접촉배양야현시NSC향종류간세포구적천이화포요,병차능구현저억제종류간세포구적생장(P＜0.05)。결론 GSC체외가추화NSC향기천이,기추화작용교분화적종류세포경위현저,병차여기분비고수평적생장인자유관;향종류간세포구천이적NSC가억제기체외생장。
Objective To observe the migration of neural stem cells (NSCs) to glioma stem cells (GSC), and to investigate the chemotactic mechanism. Methods GSCs were cultured and expanded in stem cell medium from human glioma cell line U251 and surgically resected specimens of human gliomas. All the GSCs were identified by using flow cytometry and Western blotting. Migrations of NSCs to GSCs,differentiated cells and concentration gradient fluid of growth factors were detected by Transwell assay. Concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secreted by GSCs and differentiated cells were determined by using enzyme linked immunosorbent assay (ELISA). The in vitro migration of NSCs (marked by Dio) to GSCs (marked by Dil) was observed by using fluorescent microscopy. Results Neurospheres of GSCs, with high expression of stemness markers (CD133: 11.02％-33.55％ ), were cultured in stem cell medium and could be differentiated in serumcontaining medium. After differentiation, the expression of stemness markers was decreased, while the marker of glial fibrillary acidic protein (GFAP) was increased ( P ＜ 0.05). Meanwhile, the concentrations of VEGF and bFGF secreted by GSCs were higher than those by differentiated cells. Both GSCs and differentiated cells could induce the migration of NSCs, while GSCs showed significantly stronger chemotactic effect (P ＜ 0. 05). The migratory ability of NSCs might be positively correlated with the concentration of growth factors in chemotactic fluid ( P ＜ 0. 05 ). NSCs could migrate to GSCs and surround the neurosphere of GSCs, which displays cytostatic effect in vitro. Conclusion Gliona stem cells could induce the migration of NSCs and show enhanced chemotaxis compared with the differentiated cells. This chemotactic mechanism might be related to high concentrations of growth factors secreted by GSCs. Migration of NSCs to GSCs displays cytostatic effect in vitro.