中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2013年
5期
576-583
,共8页
曹代桂%周先虎%冯世庆%陈家童%孔晓红%郝岩
曹代桂%週先虎%馮世慶%陳傢童%孔曉紅%郝巖
조대계%주선호%풍세경%진가동%공효홍%학암
小鼠%生殖细胞%许旺细胞%神经生长因子类%细胞分化
小鼠%生殖細胞%許旺細胞%神經生長因子類%細胞分化
소서%생식세포%허왕세포%신경생장인자류%세포분화
Mice%Germ cells%Schwann cells%Nerve growth factors%Cell differentiation
目的 探索小鼠胚芽干细胞(embryonic germ cells,EGCs)分离、培养的有效方法；观察激活态雪旺细胞(activated Schwann cells,ASCs)源性神经营养因子对小鼠EGCs向神经细胞分化的影响.方法 分离受精11d胚胎小鼠生殖腺嵴及同时取少量的腹壁组织共同消化培养,经胰蛋白酶消化制备成EGCs悬液,将细胞种植在饲养层细胞上.观察小鼠EGCs集落形成情况,并采用阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色对其进行鉴定.采用双酶消化组织块法结合机械分离法分离培养ASCs.神经诱导实验采用基础培养基+小鼠EGCs(空白对照组)和ASCs培养基与小鼠EGCs共培养(实验组),培养3周后对神经诱导结果进行NeuN、MBP、GFAP免疫荧光染色鉴定,计算细胞染色阳性率,对实验结果进行统计学分析.结果 小鼠EGCs呈集落性生长,集落隆起、多数呈鸟巢状、与周围饲养层细胞存在明显界限；集落内的细胞密集,呈现一个或几个核仁、核质比高的圆形或卯圆形；阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色均阳性.小鼠EGCs神经诱导1周后,倒置相差显微镜下可见EGCs克隆的边缘出现向外迁移的细胞,圆形或椭圆形,部分细胞有突起伸出.3周后,迁移的、有细长突起的细胞越来越多,NeuN、MBP、GFAP免疫荧光染色均阳性.细胞染色阳性率比较差异有统计学意义.结论 通过一种简单、经济的方法体外成功分离并获得小鼠EGCs；ASCs源性神经营养因子能够促进小鼠EGCs在体外向神经细胞分化.
目的 探索小鼠胚芽榦細胞(embryonic germ cells,EGCs)分離、培養的有效方法；觀察激活態雪旺細胞(activated Schwann cells,ASCs)源性神經營養因子對小鼠EGCs嚮神經細胞分化的影響.方法 分離受精11d胚胎小鼠生殖腺嵴及同時取少量的腹壁組織共同消化培養,經胰蛋白酶消化製備成EGCs懸液,將細胞種植在飼養層細胞上.觀察小鼠EGCs集落形成情況,併採用階段特異性胚胎抗原-1染色、堿性燐痠酶染色、過碘痠-雪伕染色對其進行鑒定.採用雙酶消化組織塊法結閤機械分離法分離培養ASCs.神經誘導實驗採用基礎培養基+小鼠EGCs(空白對照組)和ASCs培養基與小鼠EGCs共培養(實驗組),培養3週後對神經誘導結果進行NeuN、MBP、GFAP免疫熒光染色鑒定,計算細胞染色暘性率,對實驗結果進行統計學分析.結果 小鼠EGCs呈集落性生長,集落隆起、多數呈鳥巢狀、與週圍飼養層細胞存在明顯界限；集落內的細胞密集,呈現一箇或幾箇覈仁、覈質比高的圓形或卯圓形；階段特異性胚胎抗原-1染色、堿性燐痠酶染色、過碘痠-雪伕染色均暘性.小鼠EGCs神經誘導1週後,倒置相差顯微鏡下可見EGCs剋隆的邊緣齣現嚮外遷移的細胞,圓形或橢圓形,部分細胞有突起伸齣.3週後,遷移的、有細長突起的細胞越來越多,NeuN、MBP、GFAP免疫熒光染色均暘性.細胞染色暘性率比較差異有統計學意義.結論 通過一種簡單、經濟的方法體外成功分離併穫得小鼠EGCs；ASCs源性神經營養因子能夠促進小鼠EGCs在體外嚮神經細胞分化.
목적 탐색소서배아간세포(embryonic germ cells,EGCs)분리、배양적유효방법；관찰격활태설왕세포(activated Schwann cells,ASCs)원성신경영양인자대소서EGCs향신경세포분화적영향.방법 분리수정11d배태소서생식선척급동시취소량적복벽조직공동소화배양,경이단백매소화제비성EGCs현액,장세포충식재사양층세포상.관찰소서EGCs집락형성정황,병채용계단특이성배태항원-1염색、감성린산매염색、과전산-설부염색대기진행감정.채용쌍매소화조직괴법결합궤계분리법분리배양ASCs.신경유도실험채용기출배양기+소서EGCs(공백대조조)화ASCs배양기여소서EGCs공배양(실험조),배양3주후대신경유도결과진행NeuN、MBP、GFAP면역형광염색감정,계산세포염색양성솔,대실험결과진행통계학분석.결과 소서EGCs정집락성생장,집락륭기、다수정조소상、여주위사양층세포존재명현계한；집락내적세포밀집,정현일개혹궤개핵인、핵질비고적원형혹묘원형；계단특이성배태항원-1염색、감성린산매염색、과전산-설부염색균양성.소서EGCs신경유도1주후,도치상차현미경하가견EGCs극륭적변연출현향외천이적세포,원형혹타원형,부분세포유돌기신출.3주후,천이적、유세장돌기적세포월래월다,NeuN、MBP、GFAP면역형광염색균양성.세포염색양성솔비교차이유통계학의의.결론 통과일충간단、경제적방법체외성공분리병획득소서EGCs；ASCs원성신경영양인자능구촉진소서EGCs재체외향신경세포분화.
Objective To seek an optimal method for the separation,culture of mouse embryonic germ cells (EGCs) in vitro,and to observe the influence of Activated Schwann cells (ASCs)-derived neurotrophins on the differentiation capability of mouse EGCs into neurogenic cells.Methods The gonadal ridges and a few abdominal tissues of the 11-day postcoitum (dpc) mouse embryos were isolated and disaggregated by 0.125％ trypsin-0.02％ EDTA,followed by culture of the mouse EGCs on mouse embryonic fibroblast (MEF) feeders.Monoclonal formation of the mouse EGCs was observed,and the staining of stage specificity embryo antigen-1 (SSEA-1),alkaline phosphatase (AKP),periodic acid-Schiff staining (PAS) were applied to identify the mouse EGCs.Two groups were divided as followed:mouse EGCs+basic medium (control group) and mouse EGCs+ASCs (experimental group).Immunofluorescence (NeuN,MBP,GFAP)analysis was used to evaluate the neurogenic differentiation of mouse EGCs and then to calculate the statistical positive rates of cell staining.All experimental results were analyzed statistically.Results (1) Identification ofmouse EGCs:Mouse EGCs were characterized by a dome-shaped colony containing a large nucleus and a relatively small amount of cytoplasm.All mouse EGCs were positive staining of SSEA-1,AKP,and PAS;(2)The neural induction of mouse EGCs:After one week induction,there were few round or oval cells with long axon-like processes migrating from the edge of the EGCs clones.3 weeks later,the neurogenic-like cells increased quickly.The results of immunofluorescence (NeuN,MBP,GFAP)staining demonstrated that mouse EGCs could differentiate into neurogenic cells under the influence of ASCs.The positive rate of cell staining was significant.Conclusion In this study,a simple,economical method was applied to successfully separate the mouse EGCs in vitro; mouse EGCs can differentiate into neurogenic cells under the influence of ASCs-derived neurotrophins.
