摘要
背景:骨髓间充质干细胞具有向卵母细胞分化的潜能,但是其免疫排斥和来源的有限性限制了其应用。研究表明,脐带间质干细胞也具有分化为卵母样细胞的潜能。目的:建立分离培养人脐带间质干细胞的方法,观察其在体外向生殖细胞分化的潜能。方法:无菌条件下取正常足月分娩新生儿的脐带,用Ⅰ型胶原酶消化,分离单个核细胞,DMEM培养,获得贴壁细胞,流式细胞仪检测其免疫表型。采用不同条件培养基诱导细胞向成脂、成骨、成软骨方向分化。通过RT-PCR检测脐带间质干细胞中生殖系特异性标记物的表达,采用卵泡液作为条件培养基,体外诱导脐带间质干细胞向生殖细胞分化。结果与结论:①分离的脐带单核细胞培养后呈纺锤体样,体外增殖达10代以上,其分子免疫表型为:CD29、CD44、CD73(SH3)、CD90、CD105(SH2)阳性;SSEA-4弱阳性;CD31、CD34、CD45、HLA-DR阴性,与骨髓间充质干细胞的免疫表型相似。在不同的诱导条件下,脐带间质干细胞可形成脂滴、骨结节、软骨结节等结构,并表达脂肪、骨及软骨细胞相关的特异性基因。②脐带间质干细胞表达OCT4、Stella、Ifitm3等生殖系标记物,在含5%,10%,20%等不同浓度卵泡液的诱导条件下细胞可聚集分化形成卵母细胞样结构。
BACKGROUND：Bone marrow mesenchymal stem cells（BM-MSCs） have been shown to possess the potential to differentiate into oocytes.However,immune rejection and a limited number of donors of BM-MSCs constrain the applications of BM-MSCs.Several studies have demonstrated that human umbilical cord matrix stem cells（UC-MSCs） also have an intrinsic ability to differentiate into oocyte-like cells in vitro.OBJECTIVE：To establish the method for UC-MSCs culture and to investigate the in vitro differentiation potential of UC-MSCs towards germ cells.METHODS：Umbilical cord from full-term normal deliveries was obtained in sterile condition.Collagenase I-digested cells were cultured in DMEM.The immunophenotype of cells was determined by flow cytometry.Lipoblasts,osteoblasts and chondroblasts were induced in different condition cultures.The expression of germ cells specific marker in UC-MSCs was determined by reverse transcription-polymerase chain reaction.Follicular fluid was employed to induce UC-MSCs differentiation into germ cells.RESULTS AND CONCLUSION：Spindle-like umbilical cord cells were shown and cells in culture were extended to more than 10 passages.BM-MSCs-like immunophenotypes were shown：CD29,CD44,CD73（SH3）,CD90 and CD105（SH2） were positive;SSEA-4 was weakly positive;CD31,CD34,CD45 and HLA-DR were negative.After UC-MSCs were induced in different condition cultures,lipid droplet-,bone tubercle-,and cartilage tubercle-like structures emerged and the mRNA expressions of specific gene of fat,bone and cartilage were observed.Germ cells markers,OCT4,Stella,Ifitm3,were expressed in UC-MSCs.After induced by 5%,10% or 20% follicular fluid,cells aggregated and oocyte-like structures were observed.Human UC-MSCs could be cultured and amplificated in vitro.UC-MSCs showed immunophenotypes similar to BM-MSCs.UC-MSCs had the potential to differentiate into lipoblasts,osteoblasts,and chondroblasts.Oocyte-like structure was induced in vitro from UC-MSCs with germ cells specific marker.These findings suggest that UC-NSCs have the potential to differentiate into germ cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第40期7583-7587,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
supported by grants from the Scientific Research Division of Southwest Hospital
