胎盘多分化潜能细胞的体外分离培养(英文)

Culture of multipotent cells isolated from human term placenta

背景:一些研究提示,胎盘可以作为一种新的间充质干细胞来源。胎盘组织中分离出的贴壁细胞与骨髓间充质干细胞有相似形态和细胞表面标志,具有分化为成骨细胞以及神经细胞的能力。目的:探索一种新的获取间充质干细胞的来源及方法。设计:观察性实验。单位:无锡市第三人民医院。材料:胎盘(我院妇产科剖腹产手术中胎盘,经家属同意并经院伦理委员会通过签署知情同意书)。培养基;sABC试剂盒、DAB显色试剂盒;羊抗鼠-FITC;重组人碱性成纤维细胞生长因子;兔抗人神经元特异性烯醇化酶;兔抗人胶质纤维酸性蛋白。方法:实验于2005-05/2006-08在无锡第三人民医院细胞与分子生物学研究室完成。消化胎盘实体组织,贴壁培养细胞,观测形态,绘制出各代PDMCs的生长曲线。取原代培养细胞1和7d时的上清液,用化学发光法测定β-HCG含量。检测细胞表面抗原表达以及分化潜能。培养细胞在诱导分化24h(神经细胞),2周(成骨细胞)后采用常规方法免疫细胞化学染色。显色后在常规显微镜或荧光显微镜下观察。主要观察指标:①形态学以及生长曲线的观察;②多分化潜能细胞培养上清液β-HCG测定、流式细胞仪检测多分化潜能细胞抗原表达;③多分化潜能细胞诱导分化和诱导分化后细胞免疫组织化学分析。结果:①多分化潜能细胞的原代培养情况:胎盘组织消化后获得细胞中仅有少量的贴壁细胞,经两周后逐渐形成扁平单层细胞,呈漩涡状生长或成簇生长,随着细胞密度的增加,胞体变得细长,形态类似成纤维细胞。②多分化潜能细胞的生长曲线分析结果:细胞在接种后的2～8d为生长的潜伏期,细胞逐渐开始出现贴壁,无明显扩增;8d以后细胞进入对数生长期,此时细胞增殖活跃,相差显微镜下观察细胞突起向周围伸展,出现两个核细胞分裂相的间充质干细胞多见,细胞密度增大,彼此相连;11～14d,生长曲线逐渐进入平台期,MSCs铺满瓶底,细胞扩增趋缓,原代培养结束。③β-HCG测定结果:两个时间点培养上清液中均未检测到β-HCG表达。④多分化潜能细胞的表面抗原特性:多分化潜能细胞表达CD29,CD44和CD105,不表达CD34,CD45,CD19和CD106。⑤多分化潜能细胞的诱导分化结果:向神经细胞诱导24h后,细胞形态明显改变,胞体回缩,胞核部分折光性增强,出现类似于树突及轴突样结构,染色可见神经元特异性烯醇化酶、胶质纤维酸性蛋白阳性。结论:胎盘组织中含有的多分化潜能细胞与骨髓间充质干细胞形态功能相似,胎盘可以作为获取间充质干细胞的一种有效来源。

BACKGROUND： Some researches suggest that placenta is regarded as a new source of mesemchymal stem cells （MSCs）. Adherent cells derived from placenta tissue have similar morphological characteristics and surface markers to MSCs; meanwhile, they can differentiate into osteoblasts and nerve cells.OBJECTIVE： To find a new source and way for MSCs separation.DESIGN： Observational study.SETTING： The Third People＇s Hospital of Wuxi.MATERIALS： Placenta was sourced from cesarean in our department of obstetrics and gynecology and provided confirmed consent from the relatives and ethics committee. The main reagents contained culture medium, sABC kit, DAB staining kit, caprine-anti-rat FITC, recombinant human basic fibroblast growth factor （rh-bFGF）, rabbit-anti-human glial fibriliary acidic protein （GFAP）.METHODS： The experiment was carried out in the Laboratory of Cells ＆ Molecular Biology, the Third People＇s Hospital of Wuxi from May 2005 to August 2006. Placenta entity tissue was digested, adherent cells were cultured, morphological characteristics were observed, and growth curves of placenta-derived multipotent cells （PDMCs） in various generations were drawn. On the 1st and 7th days, supernatant was derived from cells in primary culture to measure content of β-glycerophosphate disodium （β-HCG） with chemiluminescence technique. In addition, expression of surface antigen and differentiating potency were detected at the same time. At 24 hours （at phase of nerve cells） and 2 weeks （at phase of osteoblasts） after inducible differentiation, cultured cells were dealt with routine immunocytochemical stain, and then they were observed under routine microscope or fluorescence microscope.MAIN OUTCOME MEASURES： ① Morphological indexes and growth curves of PDMCs; ② measurement of β-HCG in supernatant of PDMCs and expression of antigen of PDMCs with flow cytometer; ③ immunohistochemical analysis of PDMCs during and after inducible differentiation.RESULTS： ① Primary culture of PDMCs： After digestion of placenta tissue, a few of adherent cells were obtained and gradually formed thin and flat monolayer cells two weeks later. The monolayer cells grew like whirlpool or cluster. With the increase of cell density, soma was slenderer and slenderer and like fibroblast. ② Growth curves of PDMCs： Growth latency ranged from 2 to 8 days after cell inoculation. During this period, adherence was observed gradually but not obviously amplified. Eight days later, cells entered log growth phase. During this period, proliferation was active and cell process surrounding extended under phase contrast microscope. A lot of MSCs were observed in division phase between two nuclei. In addition, density was increased and cells connected to each other. Within 11-14 days, growth curve gradually entered platform phase. MSCs covered the bottom of bottle, cells slowly expanded, and primary culture stopped. ③ Measurement of β-HCG： Expression of β-HCG was not detected in supernatant at two time points. ④ Characteristics of surface antigen of PDMCs： PDMCs could express CD29, CD44 and CD105, but not CD34, CD45,CD19 and CD106. ⑤ Inducible differentiation of PDMCs： At 24 hours after inducing to nerve cells, form of PDMCs obviously changed, soma rebounded, refraction of nucleus was partially reinforced, structure was similar to dendrite and axis-cylinder, and positive neuro-specific enolase and GFAP were observed after staining.CONCLUSION：Placenta tissue contains PDMCs whose morphological function is similar to MSCs; in addition, placenta is regarded as an effective source of MSCs.