CAJ | 학술논문

背景:目前有关脐血间充质干细胞的体外培养和大规模扩增方法不一,存在一定困难.目的:观察脐血间充质样干细胞体外分离和培养的方法,并检测其表面分子的变化.方法:取新鲜采集脐带血,用1.077 g/cm3的淋巴细胞分层液,密度梯度离心法分离脐血单个核细胞.将脐血单个核细胞接种于37℃、含体积分数为5%CO2培养箱内培养.于不同时间观察细胞形态的变化并通过流式细胞仪检测细胞表面分子的表达情况.结果与结论:从脐血中分离出的单个核细胞,培养中先出现大量的造血细胞集落,CFU-GM与BFU-E集落形成最多,集落分别增加了(37.1+2.3)和(10.4+1.7)倍,瑞氏染色显示这些细胞大多数为粒系的集落(80.1±85.2)%,其次为红系的细胞集落(14.2±1.8)%,7d后出现贴壁的扁平状上皮样细胞和长梭形成纤维样细胞,同时有大量的破骨样细胞混杂.扩增后第14天经流式细胞仪分析CD38+细胞为1.64%,CD34+/CD38+细胞为1_71%,CD34+/CD38-细胞为0.55%,PI+细胞为0.05%,Annexin-V+细胞为0.18%.随着培养时间的延长,细胞数目不断增加,培养21 d时,单个核细胞扩增了近7.8倍.,第28天增加了1.71倍.经流式细胞仪分析CD38+细胞为74.32%,CD34+/CD38+细胞为1.61%,CD34+/CD38-细胞为0.24%.提示脐血间充质干细胞可以体外培养.
배경:목전유관제혈간충질간세포적체외배양화대규모확증방법불일,존재일정곤난.목적:관찰제혈간충질양간세포체외분리화배양적방법,병검측기표면분자적변화.방법:취신선채집제대혈,용1.077 g/cm3적림파세포분층액,밀도제도리심법분리제혈단개핵세포.장제혈단개핵세포접충우37℃、함체적분수위5%CO2배양상내배양.우불동시간관찰세포형태적변화병통과류식세포의검측세포표면분자적표체정황.결과여결론:종제혈중분리출적단개핵세포,배양중선출현대량적조혈세포집락,CFU-GM여BFU-E집락형성최다,집락분별증가료(37.1+2.3)화(10.4+1.7)배,서씨염색현시저사세포대다수위립계적집락(80.1±85.2)%,기차위홍계적세포집락(14.2±1.8)%,7d후출현첩벽적편평상상피양세포화장사형성섬유양세포,동시유대량적파골양세포혼잡.확증후제14천경류식세포의분석CD38+세포위1.64%,CD34+/CD38+세포위1_71%,CD34+/CD38-세포위0.55%,PI+세포위0.05%,Annexin-V+세포위0.18%.수착배양시간적연장,세포수목불단증가,배양21 d시,단개핵세포확증료근7.8배.,제28천증가료1.71배.경류식세포의분석CD38+세포위74.32%,CD34+/CD38+세포위1.61%,CD34+/CD38-세포위0.24%.제시제혈간충질간세포가이체외배양.
BACKGROUND:Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells(UCB-MSCs).OBJECTIVE:To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS:The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION:The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by(37.1±2.3)and (10.4±1.7),respectively.Switzerland staining showed most of them were granulocyte clones(80,1±85.2)%,next was erythroid clones(14.2±1.8)%.At 7 days after culture,some shuttle fibroblast-like cells and fiat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38+ cells accounted for 1.64%,and CD34+/CD38+ cells accounted for 1,71%,and CD34+/CD38- were 0.55%.PI+ and Annexin-V+ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38+,CD34+/CD38+ and CD34+/CD38- cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.