体外扩增巨核细胞的研究
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摘要
目的:探讨采用脐血单个核细胞为起始细胞在无血清培养基中体外扩增巨核细胞;观察胎儿肺部组织来源的间充质干细胞对脐血单个核细胞诱导分化为巨核细胞的影响。方法:分取脐血单个核细胞,在TPO,IL—11,肝素的扩增体系中分为两组培养,第一组以CD34~+细胞作为对照,单独培养,第二组与间充质干细胞共培养,在7天,10天,14天时,取细胞进行活细胞计数,流式细胞仪分析CD41a和CD61的表达情况。取14天细胞采用免疫组化和透射电镜分析细胞形态与结构,通过流式细胞仪分析细胞DNA含量。结果:MNC经细胞因子诱导后,CD41a和CD61阳性比例明显上升,与0天时相比,培养14天后,CD41a~+细胞平均扩增了429.5倍,CD61~+细胞平均扩增了339倍。第二组在10天时得到的CD41~+细胞和CD61~+细胞最多,分别是第一组的4.5倍和4.7倍,免疫组化和电镜结果表明,两组细胞核发育不成熟。结论:在TPO,IL—11,肝素的扩增体系下以MNC为起始细胞能够诱导得到大量的巨核细胞,胎肺间充质干细胞能有效刺激CD41~+细胞和CD61~+细胞生成,对细胞核发育影响。
Aim:In order to expand megakaryocyte from umbilical cord blood mononuclear cells,and analysis the effect of fetal lung mesenchymal stem cell(FL-MSC) on differentiation of umbilical cord blood mononuclear cells(MNC) toward megakaryocyte.Method:umbilical cord blood fresh MNC was isolated,and divided into two groups under TPO and IL-11 with heparin.First group:MNC was cultured alone and CD34~+cells were selected as control:second group: MNC was cocultured with FL-MSC.The cells were taken out at day 7,10,14 for cell counting and flow cytometry analysis CD41a and CD61.The morphous and uhrastructur of megakaryocyte,were observed by histochemis and analyzed by transmission electron microscopy at day 14.The content of DNA was analyzed at day 14. Result:After induction CD41a~+ cells and CD61~+ cells were largely increasly. At day 14,CD41a~+ cells were 429.5-fold versus initial CD41a~+ cell number at day 0 and CD61~+ cells were 339.1-fold versus initial CD61~+ cell number at day 0.The most CD41a~+ cells and CD61~+ cells were obtained at day 10 by second group,respectively 4.5 and 4.7 fold versus MNC alone.The morphous and uhrastructur of megakaryocyte showed cell nucleus of megakaryocyte were not mature both of two groups.Conclusion: MNC can generate larger amount MKs under TPO and IL-11 with heparin in serum free.FL-MSC can enhance production of CD41a~+ cells and CD61~+ cells,and had effect on cell nucleus development.
Aim:In order to expand megakaryocyte from umbilical cord blood mononuclear cells,and analysis the effect of fetal lung mesenchymal stem cell(FL-MSC) on differentiation of umbilical cord blood mononuclear cells(MNC) toward megakaryocyte.Method:umbilical cord blood fresh MNC was isolated,and divided into two groups under TPO and IL-11 with heparin.First group:MNC was cultured alone and CD34~+cells were selected as control:second group: MNC was cocultured with FL-MSC.The cells were taken out at day 7,10,14 for cell counting and flow cytometry analysis CD41a and CD61.The morphous and uhrastructur of megakaryocyte,were observed by histochemis and analyzed by transmission electron microscopy at day 14.The content of DNA was analyzed at day 14. Result:After induction CD41a~+ cells and CD61~+ cells were largely increasly. At day 14,CD41a~+ cells were 429.5-fold versus initial CD41a~+ cell number at day 0 and CD61~+ cells were 339.1-fold versus initial CD61~+ cell number at day 0.The most CD41a~+ cells and CD61~+ cells were obtained at day 10 by second group,respectively 4.5 and 4.7 fold versus MNC alone.The morphous and uhrastructur of megakaryocyte showed cell nucleus of megakaryocyte were not mature both of two groups.Conclusion: MNC can generate larger amount MKs under TPO and IL-11 with heparin in serum free.FL-MSC can enhance production of CD41a~+ cells and CD61~+ cells,and had effect on cell nucleus development.
引文
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