CAJ | 학술논문

背景:脐血间充质干细胞为干细胞领域的研究热点,但目前传代及扩增此类细胞尚无简单、有效、完美培养方法.目的:采用不同的培养基分离培养融合状态的间充质干细胞,以筛选一种较好的体外培养人脐血间充质干细胞的方法.方法:无菌条件下取正常足月剖宫产新生儿的脐血,随机分为5组:低糖DMEM(Dulbecco改良的Eagle培养基)组、高糖DMEM组、α-DMEM组、低糖DMEM+干细胞因子组、低糖DMEM+骨髓间充质干细胞培养上清组.用淋巴细胞分离液分离脐血的单个核细胞.将脐血单个核细胞接种于含体积分数为10%胎牛血清的上述培养基中,放置于37℃、体积分数为5%的CO2培养箱内培养,倒置显微镜观察细胞数量和形态的变化并用流式细胞技术分析细胞的表面抗原.结果与结论:①各组间充质干细胞培养48 h后贴壁细胞数和细胞存活率的比较:低糖DMEM+干细胞因子组、低糖DMEM+骨髓间充质干细胞培养上清组的贴壁细胞数明显多于低糖DMEM组、高糖DMEM组、α-DMEM组(P<0.05),细胞存活率亦明显高于低糖DMEM组、高糖DMEM组、α-DMEM组(P<0.05).②各组间充质干细胞在不同培养时间下生长状态的比较:培养第3,6,9,12,15,18,21天低糖 DMEM+干细胞因子组、低糖 DMEM+骨髓间充质干细胞培养上清组细胞增殖的速度均快于低糖DMEM组、高糖DMEM组、α-DMEM组(P <0.05).低糖DMEM+干细胞因子组与低糖DMEM +骨髓间充质干细胞培养上清组比较差异无显著性意义.结果可见人脐血间充质干细胞与骨髓间充质干细胞培养上清或干细胞因子共孵育,对脐血间充质干细胞体外分离培养及扩增有支持作用.
배경:제혈간충질간세포위간세포영역적연구열점,단목전전대급확증차류세포상무간단、유효、완미배양방법.목적:채용불동적배양기분리배양융합상태적간충질간세포,이사선일충교호적체외배양인제혈간충질간세포적방법.방법:무균조건하취정상족월부궁산신생인적제혈,수궤분위5조:저당DMEM(Dulbecco개량적Eagle배양기)조、고당DMEM조、α-DMEM조、저당DMEM+간세포인자조、저당DMEM+골수간충질간세포배양상청조.용림파세포분리액분리제혈적단개핵세포.장제혈단개핵세포접충우함체적분수위10%태우혈청적상술배양기중,방치우37℃、체적분수위5%적CO2배양상내배양,도치현미경관찰세포수량화형태적변화병용류식세포기술분석세포적표면항원.결과여결론:①각조간충질간세포배양48 h후첩벽세포수화세포존활솔적비교:저당DMEM+간세포인자조、저당DMEM+골수간충질간세포배양상청조적첩벽세포수명현다우저당DMEM조、고당DMEM조、α-DMEM조(P<0.05),세포존활솔역명현고우저당DMEM조、고당DMEM조、α-DMEM조(P<0.05).②각조간충질간세포재불동배양시간하생장상태적비교:배양제3,6,9,12,15,18,21천저당 DMEM+간세포인자조、저당 DMEM+골수간충질간세포배양상청조세포증식적속도균쾌우저당DMEM조、고당DMEM조、α-DMEM조(P <0.05).저당DMEM+간세포인자조여저당DMEM +골수간충질간세포배양상청조비교차이무현저성의의.결과가견인제혈간충질간세포여골수간충질간세포배양상청혹간세포인자공부육,대제혈간충질간세포체외분리배양급확증유지지작용.
BACKGROUND:The umbilical cord blood-derived mesenchymal stem cel s are the hot spot in the field of stem cel s, and there is no simple, effective culture method for the passage and amplification of umbilical cord blood-derived mesenchymal stem cel s. OBJECTIVE:To explore a better culture method of human umbilical cord blood-derived mesenchymal stem cel s in vitro with different media in the separation of mesenchymal stem cel s at a confluent status. METHODS:Human umbilical cord blood was sterilely col ected from ful-term deliveries scheduled for cesarean section. They were assigned randomly into five groups:low-glucose culture medium group, high-glucose culture medium group,α-culture medium group, low-glucose culture medium+stem cel factor group, low-glucose culture medium+human marrow mesenchymal stem cel s supernatant group. Al culture medium used was Dulbecco’s modIfied Eagle’s medium. The cord blood mononuclear cel s were isolated by lymphocyte separation medium. The monocytes of cord blood were inoculated into the culture medium containing 10%fetal bovine serum at 37 ℃ incubator with 0.05 volume fraction of CO2. Quantity and formation of cel s were observed with invert microscope, and surface antigenic features were analyzed with flow cytometry. RESULTS AND CONCLUSION:(1) Comparison on number of adherent cel s and survival rate of mesenchymal stem cel s cultured for 48 hours:Number of adherent cel s in the low-glucose culture medium+human marrow mesenchymal stem cel s supernatant group, and low-glucose culture medium+stem cel factor group were significantly increased (P<0.05), while the survival rate of cel s was also increased compared with low-glucose culture medium group, high-glucose culture medium group andα-culture medium group (P<0.05). (2) Comparison on growth status of mesenchymal stem cel s at different culture time points:Cel proliferation in the low-glucose culture medium+human marrow mesenchymal stem cel s supernatant group, and low-glucose culture medium+stem cel factor group was more rapid than that in low-glucose culture medium group, high-glucose culture medium group andα-culture medium group at 3, 6, 9, 12, 15, 18 and 21 days (P<0.05). There was no significant difference between low-glucose culture medium+human marrow mesenchymal stem cel s supernatant group and low-glucose culture medium+stem cel factor group. Experimental findings indicate that, co-culture with low-glucose culture medium+stem cel factor or human marrow mesenchymal stem cel s supernatant can promote the in vitro isolation, culture and proliferation of human umbilical cord blood-derived mesenchymal stem cel s.