中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2009年
12期
729-733,后插2
,共6页
冯建勋%腊晓林%马艳%毕晓娟%温浩
馮建勛%臘曉林%馬豔%畢曉娟%溫浩
풍건훈%석효림%마염%필효연%온호
羊水细胞%间充质干细胞%转录因子Oct-4%神经元
羊水細胞%間充質榦細胞%轉錄因子Oct-4%神經元
양수세포%간충질간세포%전록인자Oct-4%신경원
Amniotic fluid cell%Mesenchymal stem cell%Transcription factor Oct-4%Neuron
目的 建立两步培养法分离培养人孕中期羊水间充质干细胞(MSCs)的方法,观察羊水MSCs向神经元样细胞的诱导分化.方法 采用改进的两步培养法分离和培养人羊水MSCs,以β-巯基乙醇诱导其向神经元样细胞方向分化;并使用流式细胞术、逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学法研究人羊水MSCs的生物学特性.结果 成功分离、培养和传代出人羊水MSCs.流式细胞术检测人羊水MSCs表达CD44、CD29、CD105,不表达CD45、CD34、人白细胞DR抗原(HLA-DR);免疫荧光法和RT-PCR检测表明,部分人羊水MSCs表达转录因子Oct-4.两步培养法羊水MSCs集落形成率[(15.0±2.3)%]和Oct-4阳性细胞率[(1.2±0.3)%]均高于一步法[分别为(10.0±1.8)%,(0.9±0.2)%,P均<0.05].羊水MSCs经β-巯基乙醇诱导后细胞形态发生改变,伸出突起并呈神经球样改变;免疫细胞化学法检测显示(54.76±3.65)%的细胞表达神经元特异性烯醇化酶(NSE),同时(36.28±4.27)%的细胞表达神经胶质酸性蛋白(GFAP).结论 人羊水中存在MSCs,两步培养法是一种高效、简便、实用而且不干扰常规产前诊断程序的方法.在体外条件下,利用β-巯基乙醇和适宜的培养液可使羊水MSCs定向分化为神经元.
目的 建立兩步培養法分離培養人孕中期羊水間充質榦細胞(MSCs)的方法,觀察羊水MSCs嚮神經元樣細胞的誘導分化.方法 採用改進的兩步培養法分離和培養人羊水MSCs,以β-巰基乙醇誘導其嚮神經元樣細胞方嚮分化;併使用流式細胞術、逆轉錄-聚閤酶鏈反應(RT-PCR)和免疫細胞化學法研究人羊水MSCs的生物學特性.結果 成功分離、培養和傳代齣人羊水MSCs.流式細胞術檢測人羊水MSCs錶達CD44、CD29、CD105,不錶達CD45、CD34、人白細胞DR抗原(HLA-DR);免疫熒光法和RT-PCR檢測錶明,部分人羊水MSCs錶達轉錄因子Oct-4.兩步培養法羊水MSCs集落形成率[(15.0±2.3)%]和Oct-4暘性細胞率[(1.2±0.3)%]均高于一步法[分彆為(10.0±1.8)%,(0.9±0.2)%,P均<0.05].羊水MSCs經β-巰基乙醇誘導後細胞形態髮生改變,伸齣突起併呈神經毬樣改變;免疫細胞化學法檢測顯示(54.76±3.65)%的細胞錶達神經元特異性烯醇化酶(NSE),同時(36.28±4.27)%的細胞錶達神經膠質痠性蛋白(GFAP).結論 人羊水中存在MSCs,兩步培養法是一種高效、簡便、實用而且不榦擾常規產前診斷程序的方法.在體外條件下,利用β-巰基乙醇和適宜的培養液可使羊水MSCs定嚮分化為神經元.
목적 건립량보배양법분리배양인잉중기양수간충질간세포(MSCs)적방법,관찰양수MSCs향신경원양세포적유도분화.방법 채용개진적량보배양법분리화배양인양수MSCs,이β-구기을순유도기향신경원양세포방향분화;병사용류식세포술、역전록-취합매련반응(RT-PCR)화면역세포화학법연구인양수MSCs적생물학특성.결과 성공분리、배양화전대출인양수MSCs.류식세포술검측인양수MSCs표체CD44、CD29、CD105,불표체CD45、CD34、인백세포DR항원(HLA-DR);면역형광법화RT-PCR검측표명,부분인양수MSCs표체전록인자Oct-4.량보배양법양수MSCs집락형성솔[(15.0±2.3)%]화Oct-4양성세포솔[(1.2±0.3)%]균고우일보법[분별위(10.0±1.8)%,(0.9±0.2)%,P균<0.05].양수MSCs경β-구기을순유도후세포형태발생개변,신출돌기병정신경구양개변;면역세포화학법검측현시(54.76±3.65)%적세포표체신경원특이성희순화매(NSE),동시(36.28±4.27)%적세포표체신경효질산성단백(GFAP).결론 인양수중존재MSCs,량보배양법시일충고효、간편、실용이차불간우상규산전진단정서적방법.재체외조건하,이용β-구기을순화괄의적배양액가사양수MSCs정향분화위신경원.
Objective To isolate mesenchymal stem cells (MSCs) from second-trimester amniotic fluid using an improved two-stage culture protocol, and to induce these MSCs into neuron-like cells. Methods An improved two-stage culture protocol for MSCs from amniotic fluid was developed. MSCs from amniotic fluid were induced to differentiate with β-mercaptoethanol into neuron-like cells. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were employed for analysis of the phenotypic characteristics of the cultured MSCs from amniotic fluid. Results MSCs from amniotic fluid were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analysis showed that they were positive for CD44, CD29, and CD105, but negative for CD45, CD34, or human leucocyte antigen-DR (HLA-DR). Importantly, a subpopulation of transcription factor Oct-4 positive cells could be detectable in the cultured MSCs from amniotic fluid. Moreover, MSCs from amniotic fluid obtained by two-stage culture protocol showed higher proliferation rate [(15.0±2.3)% vs. (10.0±1.8)%] and higher Oct-4 positive cell rate [(1.2±0.3)% vs. (0.9±0.2)%, both P<0.05]. After the induction, MSCs from amniotic fluid displayed processes that formed extensive networks. Positive cells for neurone specific enolase (NSE) constituted (54.76±3.65)%, and glial fibrillary acidic protein (GFAP) (36.28±4.27)%, respectively. Conclusion We demonstrate that human pluripotent MSCs are present in second-trimester amniotic fluid. The two-stage culture protocol could be a kind of simple one with high performance and it does not interfere with the routine fetal karyotyping. MSCs from amniotic fluid can be induced to differentiate into neuron-like cells in vitro by β-mercaptoethanol in optimal medium.
