中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
45期
8854-8860
,共7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%Oct-4
羊水細胞%間充質榦細胞%Oct-4
양수세포%간충질간세포%Oct-4
背景:目前对干细胞的体外分离纯化技术大多是建立在对细胞表面标记识别的基础之上,包括单抗贴壁铺展法、流式细胞分选法、免疫磁珠分选法等,但操作复杂,价格昂贵.目的:观察两步法分离和培养人羊水来源胚胎间充质干细胞的生物学特征.设计、时间及地点:开放性实验,于2008-03/2009-03在新疆医科大学干细胞研究室完成.材料:10例羊水标本来自怀孕16～22周行产前诊断或自愿引产的孕妇,在超声引导下行羊膜腔穿刺取得20～40 mL羊水.方法:采用改进的两步培养法分离和培养人羊水间充质干细胞,首先同一步法将羊水标本离心、接种、培养至出现梭形细胞为主、类成纤维细胞的间充质干细胞集落.然后将上清培养液转移至新的25 cm~2培养瓶中继续培养,至出现梭形细胞为主、类成纤维细胞的间充质干细胞集落且达70%汇合后消化,改用α-MEM培养基,同样添加碱性成纤维细胞生长因子,接种培养,记为第1代.主要观察指标:①原代及传代羊水间充质干细胞形态学变化.②羊水间充质干细胞细胞核型、细胞周期、生长曲线和集落形成能力测定.③流式细胞仪、免疫荧光和RT-PCR检测表面抗原和细胞因子.结果:实验成功分离、培养和传代人羊水间充质干细胞.①孕中期羊水细胞原代培养平均7 d可见散在的梭形细胞为主、类成纤维细胞的间充质干细胞集落;传代细胞在12 h内完全贴壁,经过一两天潜伏期后增殖迅速,培养六七天可达90%融合,细胞排列有一定的方向性,呈漩涡状、辐射状排列,细胞为长梭形,相互之间界限不清.②两种方法得到的羊水间充质干细胞染色体核型为正常二倍体核型;生长曲线均呈S形,但两步法在第(6.1±0.5)天就达到高峰,显著快于一步法第(7.2±0.6)天(P=0.035).流式细胞仪检测结果显示两步法羊水P3细胞S期细胞占(14±2.3)%,显著多于一步法(9.0±1.4)%(P=0.031).两种方法获得的羊水间充质干细胞低密度种植7 d后,均可形成散在的细胞集落,但两步法集落形成率为(15.0±2.3)%,显著多于一步法(10.0±1.8)%(P=0.021).③流式细胞仪检测结果显示,羊水间充质干细胞表达CD44、CD29、CD105,而CD34、CD45、HLA-DR阴性;免疫荧光检测表明羊水中含有Oct-4阳性细胞,但两步法羊水间充质干细胞中Oct-4阳性细胞比例为(1.2±0.3)%,显著高于一步法(0.9±0.2)%(P=0.041);RT-PCR分析表明两种方法获得的羊水间充质干细胞均表达Oct-4.结论:人羊水中也存在间充质干细胞,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.
揹景:目前對榦細胞的體外分離純化技術大多是建立在對細胞錶麵標記識彆的基礎之上,包括單抗貼壁鋪展法、流式細胞分選法、免疫磁珠分選法等,但操作複雜,價格昂貴.目的:觀察兩步法分離和培養人羊水來源胚胎間充質榦細胞的生物學特徵.設計、時間及地點:開放性實驗,于2008-03/2009-03在新疆醫科大學榦細胞研究室完成.材料:10例羊水標本來自懷孕16～22週行產前診斷或自願引產的孕婦,在超聲引導下行羊膜腔穿刺取得20～40 mL羊水.方法:採用改進的兩步培養法分離和培養人羊水間充質榦細胞,首先同一步法將羊水標本離心、接種、培養至齣現梭形細胞為主、類成纖維細胞的間充質榦細胞集落.然後將上清培養液轉移至新的25 cm~2培養瓶中繼續培養,至齣現梭形細胞為主、類成纖維細胞的間充質榦細胞集落且達70%彙閤後消化,改用α-MEM培養基,同樣添加堿性成纖維細胞生長因子,接種培養,記為第1代.主要觀察指標:①原代及傳代羊水間充質榦細胞形態學變化.②羊水間充質榦細胞細胞覈型、細胞週期、生長麯線和集落形成能力測定.③流式細胞儀、免疫熒光和RT-PCR檢測錶麵抗原和細胞因子.結果:實驗成功分離、培養和傳代人羊水間充質榦細胞.①孕中期羊水細胞原代培養平均7 d可見散在的梭形細胞為主、類成纖維細胞的間充質榦細胞集落;傳代細胞在12 h內完全貼壁,經過一兩天潛伏期後增殖迅速,培養六七天可達90%融閤,細胞排列有一定的方嚮性,呈漩渦狀、輻射狀排列,細胞為長梭形,相互之間界限不清.②兩種方法得到的羊水間充質榦細胞染色體覈型為正常二倍體覈型;生長麯線均呈S形,但兩步法在第(6.1±0.5)天就達到高峰,顯著快于一步法第(7.2±0.6)天(P=0.035).流式細胞儀檢測結果顯示兩步法羊水P3細胞S期細胞佔(14±2.3)%,顯著多于一步法(9.0±1.4)%(P=0.031).兩種方法穫得的羊水間充質榦細胞低密度種植7 d後,均可形成散在的細胞集落,但兩步法集落形成率為(15.0±2.3)%,顯著多于一步法(10.0±1.8)%(P=0.021).③流式細胞儀檢測結果顯示,羊水間充質榦細胞錶達CD44、CD29、CD105,而CD34、CD45、HLA-DR陰性;免疫熒光檢測錶明羊水中含有Oct-4暘性細胞,但兩步法羊水間充質榦細胞中Oct-4暘性細胞比例為(1.2±0.3)%,顯著高于一步法(0.9±0.2)%(P=0.041);RT-PCR分析錶明兩種方法穫得的羊水間充質榦細胞均錶達Oct-4.結論:人羊水中也存在間充質榦細胞,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.
배경:목전대간세포적체외분리순화기술대다시건립재대세포표면표기식별적기출지상,포괄단항첩벽포전법、류식세포분선법、면역자주분선법등,단조작복잡,개격앙귀.목적:관찰량보법분리화배양인양수래원배태간충질간세포적생물학특정.설계、시간급지점:개방성실험,우2008-03/2009-03재신강의과대학간세포연구실완성.재료:10례양수표본래자부잉16～22주행산전진단혹자원인산적잉부,재초성인도하행양막강천자취득20～40 mL양수.방법:채용개진적량보배양법분리화배양인양수간충질간세포,수선동일보법장양수표본리심、접충、배양지출현사형세포위주、류성섬유세포적간충질간세포집락.연후장상청배양액전이지신적25 cm~2배양병중계속배양,지출현사형세포위주、류성섬유세포적간충질간세포집락차체70%회합후소화,개용α-MEM배양기,동양첨가감성성섬유세포생장인자,접충배양,기위제1대.주요관찰지표:①원대급전대양수간충질간세포형태학변화.②양수간충질간세포세포핵형、세포주기、생장곡선화집락형성능력측정.③류식세포의、면역형광화RT-PCR검측표면항원화세포인자.결과:실험성공분리、배양화전대인양수간충질간세포.①잉중기양수세포원대배양평균7 d가견산재적사형세포위주、류성섬유세포적간충질간세포집락;전대세포재12 h내완전첩벽,경과일량천잠복기후증식신속,배양륙칠천가체90%융합,세포배렬유일정적방향성,정선와상、복사상배렬,세포위장사형,상호지간계한불청.②량충방법득도적양수간충질간세포염색체핵형위정상이배체핵형;생장곡선균정S형,단량보법재제(6.1±0.5)천취체도고봉,현저쾌우일보법제(7.2±0.6)천(P=0.035).류식세포의검측결과현시량보법양수P3세포S기세포점(14±2.3)%,현저다우일보법(9.0±1.4)%(P=0.031).량충방법획득적양수간충질간세포저밀도충식7 d후,균가형성산재적세포집락,단량보법집락형성솔위(15.0±2.3)%,현저다우일보법(10.0±1.8)%(P=0.021).③류식세포의검측결과현시,양수간충질간세포표체CD44、CD29、CD105,이CD34、CD45、HLA-DR음성;면역형광검측표명양수중함유Oct-4양성세포,단량보법양수간충질간세포중Oct-4양성세포비례위(1.2±0.3)%,현저고우일보법(0.9±0.2)%(P=0.041);RT-PCR분석표명량충방법획득적양수간충질간세포균표체Oct-4.결론:인양수중야존재간충질간세포,량보배양법시일충고효、간편실용이차불간우상규산전진단정서적방법.
BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.
