建立鸡EPGCs单细胞克隆株初探

目的探索鸡EPGCs单细胞克隆建立细胞系的可能性,并对其生物学特性进行鉴定。方法采取19期和28期的鸡EPGCs,体外培养传至第4代的细胞聚合体,用胰酶消化将细胞分散成单细胞悬液,将单个细胞接种至96孔板,每个孔内接种1个细胞,生长出的细胞集落用胶原酶消化传代,细胞化学法和免疫荧光法检测多向分化细胞的表面标志物;常规染色体核型检查。结果接种的288个单细胞中有9个扩增,其中7个传至第2代,2个传至第4代,克隆形成率为3.1%。扩增出的2~4代单细胞克隆能稳定增殖不分化,具有正常二倍性染色体核型、碱性磷酸酶阳性、阶段特异性胚胎抗原(SSEA)-1等特征性细胞表面标志物呈阳性;离体情况下具有形成类胚体和类上皮样细胞的能力。结论建立鸡EPGCs单细胞克隆细胞系是可行的,其生物学特性稳定。

Establishment of Single-Cell Clonal Lines from Chicken EPGCs

Objective The aim of this study was to explore the possibility to establish single-cell colonal lines from chicken embryonic primordial germ cells(EPGCs) and investigate their biological characteristics.Method single-cells were derived from the 4th passages and multiplied clonal EPGCs at stage 19 and 28,which were digested with 0.25% trypsin-EDTA.Then the single cell was picked up and seeded into individual well of a 96-well plate containing fibroblast feeder-layer,and every well contained one cell.The outgrowth of single-cell clones were passed after treatment with trypsin-EDTA,and their biological characteristics were detected by cytochemistry and histoimmunochemistry to detect their special markers.The karyotypes were assessed by routine test.The pluripotency was analyzed by differentiation in vitro.Results Nine in total of 288 clones maintained the normal diploid karyotype and undifferentiated.7 clones were passaged for 2 passages,and 2 clones were passaged for 4 passages.The 9 clones possessed normal karyotypes,expressing a series of surface markers such as: alkaline phosphatase and stage-specific embryonic antigen(SSEA)-1.Conclusion Nine single cell-cloned of chicken embryonic PGCs have been derived in our laboratory.The cells possess stable biological characteristics of undifferentiated PGCs.