CAJ | 학술논문

背景:和骨髓间充质干细胞相比,脐血间充质干细胞取材方便,其免疫原性较低,能耐受更大程度卜的HLA配型不符,分离出的间充质干细胞纯度更高.目的:分析新生儿脐血间充质干细胞体外分离、纯化、扩增,以及向成骨及脂肪细胞定向诱导分化的方法与条件.设计、时间及地点:观察性实验,于2004-09/2005-11在四川大学华西医院生物治疗国家重点实验室,干细胞与组织工程研究室完成.材料:胎龄37～40周的新生儿脐血.方法:以Ficoll-HyPaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34+免疫磁珠负选法分离脐血单个核细胞.将分离获得的单个核细胞采用L-DMEM培养基+体积分数0.1胎牛血清或Mesencult TM 培养基+体积分数0.1胎牛血清进行间充质干细胞培养传代,获得的第3代集落牛长细胞进行流式细胞仪表面抗原测定,并诱导其向成骨、脂肪细胞分化.主要观察指标:①脐血间充质干细胞的鉴定.②经茜素红染色及油红染色证实其诱导分化.结果:经沉降红细胞后密度梯度离心分离的单个核细胞,使用Mesencult TM培养基+体积分数0.1胎牛血清培养成功率高,第3代可出现明显的集落生长,而另两种方法分离培养的细胞则难以形成集落;集落细胞表面抗原测定表达CD29,CD59,D71,不表达CD34,CD45及HLA-DR等分子.成骨定向诱导分化的集落细胞经茜素红染色胞浆中出现有大量的钙沉积,成脂肪定向诱导分化的集落细胞油红染色示胞浆充满油滴空熔泡.结论:使片j MesencultTM培养基+体积分数0.1胎牛血清培养由甲基纤维素沉降脐血红细胞后密度梯度离心分离的单个核细胞培养成功率高,第3代可出现明显的集落生长.经诱导后可分化为成骨细胞和脂肪细胞.
배경:화골수간충질간세포상비,제혈간충질간세포취재방편,기면역원성교저,능내수경대정도복적HLA배형불부,분리출적간충질간세포순도경고.목적:분석신생인제혈간충질간세포체외분리、순화、확증,이급향성골급지방세포정향유도분화적방법여조건.설계、시간급지점:관찰성실험,우2004-09/2005-11재사천대학화서의원생물치료국가중점실험실,간세포여조직공정연구실완성.재료:태령37～40주적신생인제혈.방법:이Ficoll-HyPaque림파세포분리액밀도제도법、침강홍세포후밀도제도법급CD34+면역자주부선법분리제혈단개핵세포.장분리획득적단개핵세포채용L-DMEM배양기+체적분수0.1태우혈청혹Mesencult TM 배양기+체적분수0.1태우혈청진행간충질간세포배양전대,획득적제3대집락우장세포진행류식세포의표면항원측정,병유도기향성골、지방세포분화.주요관찰지표:①제혈간충질간세포적감정.②경천소홍염색급유홍염색증실기유도분화.결과:경침강홍세포후밀도제도리심분리적단개핵세포,사용Mesencult TM배양기+체적분수0.1태우혈청배양성공솔고,제3대가출현명현적집락생장,이령량충방법분리배양적세포칙난이형성집락;집락세포표면항원측정표체CD29,CD59,D71,불표체CD34,CD45급HLA-DR등분자.성골정향유도분화적집락세포경천소홍염색포장중출현유대량적개침적,성지방정향유도분화적집락세포유홍염색시포장충만유적공용포.결론:사편j MesencultTM배양기+체적분수0.1태우혈청배양유갑기섬유소침강제혈홍세포후밀도제도리심분리적단개핵세포배양성공솔고,제3대가출현명현적집락생장.경유도후가분화위성골세포화지방세포.
BACKGROUND: Compared with bone marrow-derived mesenchymal stem cells (MSCs),human umbilical cord blood (hUCB)-MSCs possess many advantages,including easy acquirement,low immunogenicity,able to tolerance a higher degree of HLA-matching inconsistency,and with higher purity.OBJECTIVE: To analyze the method and condition for in vitro isolation,purification,proliferation,and osteoblast and lipoblast differentiation of hUCB-MSCs.DESIGN,TIME AND SETTING: The present observational experiment was performed in the Key National Laboratory of Biological Treatment,Huaxi Hospital,Sichuan University (i.e.,Institute of Stem Cells and Tissue EngIneering) between September 2004 and November 2005.MATERIALS: Neonatal cord blood at gestational age 37 to 40 weeks.METHODS: hUCB-MSCs were collected in aseptic condition,isolated by density gradient centrifugation,sedimenting red cells with methylcellulose followed by density gradient centrifugation,or immunomagnetic beads negative technique of CD34+.Theisolated MSCs were used to carry on plastic adherent culture in L-DMEM +10% fetal bovine serum (FBS) or MesencultTM medium +10% FBS.The third passage of cells were used for surface antigen determination by flow cytometry and induced to differentiate towards osteoblasts and lipoblasts.MAIN OUTCOME MEASURES: ①Identification of hUCB-MSCs.②Confirmation of hUCB-MSC differentiation by alizarin red and oil red staining.RESULTS: The mononuclear cells isolated by sedimented and centrifuged way cultured in MesencultTM medium +10% FBS were most available.Obvious colonies appeared in the third passage.The cells obtained by only centrifugation in density gradient were hard to form colony,and those isolated by immunomagnetic beads were hard to culture.The surface antigens of these colony cells presented CD29,CD59,and CD71,hut did not express CD34,CD45,and HLA-DR,and so on.After alizarin red staining,osteoblast-differentiating colony cell cytoplasm exhibited mineralized matrices.After old red staining,lipoblast-differentiating colony cell cytoplasm was full of lipid vacuoles.CONCLUSION: The MSCs can be successfully isolated by sedimenting red cells with methylcellulose followed by centrifugation and cultured in MesencultTM medium +10% FBS.Obvious colony growth appears in the third passage.After induction,the MSCs can differentiate into osteoblasts and lipoblasts.