中华显微外科杂志
中華顯微外科雜誌
중화현미외과잡지
Chinese Journal of Microsurgery
2010年
3期
221-223,封3
,共4页
邓许勇%闫慧博%鲁凯伍%金大地
鄧許勇%閆慧博%魯凱伍%金大地
산허용%염혜박%로개오%금대지
人脐带血%间充质干细胞%氯化锂%神经元%分化
人臍帶血%間充質榦細胞%氯化鋰%神經元%分化
인제대혈%간충질간세포%록화리%신경원%분화
Human umbilical cord blood%Mesenchymal stem cells%Lithium chloride%Neuion%Differentiation
目的 探讨氯化锂体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的可行性.方法 无菌条件下收集正常足月儿的脐带血,肝素抗凝,密度梯度离心法分离人脐血单个核细胞,贴壁法纯化,用含15%FBS的低糖DMEM培养基进行扩增培养,流式细胞仪检测表面抗原.取3代的脐血MSCs进行诱导,A组:用含15%FBS和20 ng/ml bFGF的DMEM完全培养基预诱导24 h,3 mol/L LiCI的DMEM培养基继续诱导6 d:B组:含3 mol/L LiCI的DMEM培养基诱导7 d;C组:含15%FBS的DMEM培养基正常培养7 d.光镜下观察细胞形态,用免疫组化技术检测细胞NSE、MAP2及GFAP的表达.结果 A组与B组诱导3 d后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起.免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能表达神经元特异性标志NSE和MAP2,阳性表达率A组[分别为(73.6±7.8)%,(75.5±8.5)%]明显高于B组[分别为(31.0±4.3)%、(33.5±5.O)%],而星形胶质细胞特异性标志GFAP阳性细胞较少,A、B、C三组阳性表达率分别为(4.7 ±3.3)%、(5.1±4.6)%、(8.5±3.2)%.结论 LiCI联合生长因子bFGF体外可诱导人脐血MSCs分化为神经元样细胞.
目的 探討氯化鋰體外誘導人臍血間充質榦細胞(MSCs)嚮神經細胞分化的可行性.方法 無菌條件下收集正常足月兒的臍帶血,肝素抗凝,密度梯度離心法分離人臍血單箇覈細胞,貼壁法純化,用含15%FBS的低糖DMEM培養基進行擴增培養,流式細胞儀檢測錶麵抗原.取3代的臍血MSCs進行誘導,A組:用含15%FBS和20 ng/ml bFGF的DMEM完全培養基預誘導24 h,3 mol/L LiCI的DMEM培養基繼續誘導6 d:B組:含3 mol/L LiCI的DMEM培養基誘導7 d;C組:含15%FBS的DMEM培養基正常培養7 d.光鏡下觀察細胞形態,用免疫組化技術檢測細胞NSE、MAP2及GFAP的錶達.結果 A組與B組誘導3 d後細胞即齣現形態學上的改變,細胞變成不規則形,立體感增彊,從胞體伸齣突起.免疫組織化學和免疫熒光方法鑒定顯示,誘導後的細胞能錶達神經元特異性標誌NSE和MAP2,暘性錶達率A組[分彆為(73.6±7.8)%,(75.5±8.5)%]明顯高于B組[分彆為(31.0±4.3)%、(33.5±5.O)%],而星形膠質細胞特異性標誌GFAP暘性細胞較少,A、B、C三組暘性錶達率分彆為(4.7 ±3.3)%、(5.1±4.6)%、(8.5±3.2)%.結論 LiCI聯閤生長因子bFGF體外可誘導人臍血MSCs分化為神經元樣細胞.
목적 탐토록화리체외유도인제혈간충질간세포(MSCs)향신경세포분화적가행성.방법 무균조건하수집정상족월인적제대혈,간소항응,밀도제도리심법분리인제혈단개핵세포,첩벽법순화,용함15%FBS적저당DMEM배양기진행확증배양,류식세포의검측표면항원.취3대적제혈MSCs진행유도,A조:용함15%FBS화20 ng/ml bFGF적DMEM완전배양기예유도24 h,3 mol/L LiCI적DMEM배양기계속유도6 d:B조:함3 mol/L LiCI적DMEM배양기유도7 d;C조:함15%FBS적DMEM배양기정상배양7 d.광경하관찰세포형태,용면역조화기술검측세포NSE、MAP2급GFAP적표체.결과 A조여B조유도3 d후세포즉출현형태학상적개변,세포변성불규칙형,입체감증강,종포체신출돌기.면역조직화학화면역형광방법감정현시,유도후적세포능표체신경원특이성표지NSE화MAP2,양성표체솔A조[분별위(73.6±7.8)%,(75.5±8.5)%]명현고우B조[분별위(31.0±4.3)%、(33.5±5.O)%],이성형효질세포특이성표지GFAP양성세포교소,A、B、C삼조양성표체솔분별위(4.7 ±3.3)%、(5.1±4.6)%、(8.5±3.2)%.결론 LiCI연합생장인자bFGF체외가유도인제혈MSCs분화위신경원양세포.
Objective To study the possibility of inducing human umbilical cord blood mesenchymal stem cells(MSCs) to differentiate into neuron-like cells by lithium chloride(LiCl) in vitro.Methods Human umbilical cord blood was collected from mature neonates.All samples were obtained sterilely with 20 U/ml heparin.The cord mononuclear cells were isolated with lymphocyte separation medium(density 1.077 g/ml), then purified by wall sticking screening and expanded with slight sugar DIEM containing 15% FBS.The third passage of the expanded MSCs were pre-inducted with DIEM containing 15% FBS and 20 ng/ml bFGF for 24 hours, then induced with DIEM without serum but 3 mol/L LiCl for 6 days in group A.The MSCs were induced with DIEM containing 3 mol/L Licl for 7 days in group B.The MSCs were normally cultured with DIEM containing 15% FBS in group C.The morphological changes of the cells were observed under phase contrast microscope.The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.Results After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology.The expression of NSE and MAP2 were positive for the majority cells in group A and B, and that of group A[(73.6 ± 7.8)%, 75.5 ± 8.5)% respectively]were obviously higher than group B[(31.0 ± 4.3)%,(33.5 ± 5.0)% respectively], few expressed GFAP in both groups.Conclusion The combination of LiCl and growth factor may induce the human umbilical cord blood MSCs into neuron-like cells in vitro.
