CAJ | 학술논문

目的对比研究人脐血和骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)体外的分离、培养和生物学特性,并观察其分化潜能和形态学变化,为组织工程选取种子细胞提供实验依据.方法 Ficoll密度梯度离心结合贴壁培养法分别分离纯化成人骨髓和脐血源MSCs,体外培养和连续传代,并在含有2%B27的Neurobasal培养基中添加碱性成纤维细胞生长因子、表皮生长因子,将获得的MSCs向神经干细胞定向诱导,利用倒置显微镜连续观察细胞培养、传代和向神经细胞表型转化过程的形态学变化;采用免疫组化和荧光免疫组化法对诱导后细胞进行鉴定.结果原代分离的骨髓间充质干细胞(BMSCs)在接种后48 h贴壁,7 d细胞呈长梭形,有一定的方向性,并达到90%融合;而脐血间充质干细胞(UMSCs)48 h后贴壁,似乎贴的不牢,持续14d才能形成小丛、小簇、小集落,21 d排列才有一定的方向性.培养基添加神经营养因子诱导后的细胞呈现典型的神经前体细胞样表型,免疫组化和免疫荧光结果显示,诱导后的细胞能特异性表达神经元特异性标志β微管蛋白(β-tubulin)和星形胶质细胞特异性标志神经胶质相关蛋白(GFAP).结论人脐血和骨髓中含MSCs,且具备其基本恃征,体外培养UMSCs生长速度比BMSCs缓慢10 d～15 d左右,传代以后的各组细胞生长速度与形态无明显差异.骨髓和脐血来源的MSCs在体外可定向诱导分化为神经细胞.
목적 대비연구인제혈화골수래원적간충질간세포(mesenchymal stem cells,MSCs)체외적분리、배양화생물학특성,병관찰기분화잠능화형태학변화,위조직공정선취충자세포제공실험의거.방법 Ficoll밀도제도리심결합첩벽배양법분별분리순화성인골수화제혈원MSCs,체외배양화련속전대,병재함유2%B27적Neurobasal배양기중첨가감성성섬유세포생장인자、표피생장인자,장획득적MSCs향신경간세포정향유도,이용도치현미경련속관찰세포배양、전대화향신경세포표형전화과정적형태학변화;채용면역조화화형광면역조화법대유도후세포진행감정.결과 원대분리적골수간충질간세포(BMSCs)재접충후48 h첩벽,7 d세포정장사형,유일정적방향성,병체도90%융합;이제혈간충질간세포(UMSCs)48 h후첩벽,사호첩적불뢰,지속14d재능형성소총、소족、소집락,21 d배렬재유일정적방향성.배양기첨가신경영양인자유도후적세포정현전형적신경전체세포양표형,면역조화화면역형광결과현시,유도후적세포능특이성표체신경원특이성표지β미관단백(β-tubulin)화성형효질세포특이성표지신경효질상관단백(GFAP).결론 인제혈화골수중함MSCs,차구비기기본시정,체외배양UMSCs생장속도비BMSCs완만10 d～15 d좌우,전대이후적각조세포생장속도여형태무명현차이.골수화제혈래원적MSCs재체외가정향유도분화위신경세포.
Objective To isolate and cultive mesenchymal stem cells(MSCs)derived from human umbilical cord blood and bone marrow in vitro and to investigate its basic biological characteristics,phenotype characteristics and multilineage potential.Methods MSCs derived from human umbilical cord blood and bone marrow were isolated and purified by modified ficoll density gradient centrifugation combined with adherence culture method.After expended and digestion-controlled passage,the subculture were expanded and purified.Subculture differentiation were induced into neuronlike cells by cytokine. Morphology and biological characteristics of bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. The surface molecule expressions of cells were examined by immunocytochemistry and immunocyte-fluorescence.Results 48-hour culture,BMSCs had begun to adhere. At 7 days,cell were seen adherent 90%the Wall of the culture flask.After 48-hour culture,UMSCs had begun to adhere,not fastness. At 14 days,Cell were seen accumulated colony.At 21 days,cells were gradually showed bostrychoid or whirlpool arranged parallelly. MSCs were induced to differentiate into neuron-like cells.The induced cells were monitored with immunohistochemistry and immunoeyte-fluorescence.Conclusion Human umbilical cord blood and bone marrow have MSCs,and possess the embryonic stem cell dike biological characteristics.The growth rate of UMSCs is slower than BMSCs between 10 days and 15 days.MSCs derived from human umbilical cord blood and bone marrow in vitro may differentiate into neuron-like cells.