羊水来源干细胞的生物学特性分析及其向肝细胞诱导分化的实验研究
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摘要
羊水来源干细胞(amniotic fluid-derived stem cells,AFSCs)是近几年来倍受关注的新成体干细胞类型,有关羊水干细胞的分离培养、生物学特性分析、体内外分化潜能等仍有很多问题需深入探讨。在其诱导分化方向和应用领域方面,我们针对我国肝病的发病形式和目前肝脏再生修复治疗的限制性因素,以分离、培养和鉴定羊水来源干细胞、并将其在体内外诱导分化为肝细胞为目标,建立相应的技术方法,为人类羊水来源干细胞的基础研究和临床应用奠定基础。
本研究主要包括三部分:
一、AFSCs分离培养方法的建立及生物学特性分析:
孕中期羊水(10-20ml)离心后细胞沉淀分别在下列四种不同条件下进行培养:低糖DMEM培养基(LD)+10%胎牛血清(FBS)、LD+20%胎牛血清、LD+15%胎牛血清+4ng/ml碱性成纤维细胞生长因子(bFGF)、LD+10%胎牛血清+0.1%明胶铺板。比较不同培养条件下原代集落数、可传代次数、扩增细胞数等;选择最佳条件对羊水来源干细胞进行扩增,通过形态学观察、细胞周期和生长曲线、特异性标志物检测、体外诱导分化、染色体核型分析及致瘤实验等对其生物学特性进行系统分析。
结果:15%血清结合bFGF可促进原代羊水细胞的贴壁生长和持续扩增,在此培养条件下AFSCs生长旺盛,持续传代;表达大部分间充质来源标志如CD105、CD44、CD29,以及部分全能干细胞标志如SSES-4、Oct-4和Nanog;经不同诱导分化条件培养后,从基因表达或表型上证实可向脂肪细胞、神经元及成骨细胞等不同胚层的目的细胞分化;长期培养中染色体核型稳定,注射裸鼠体内后8周未见肿瘤形成。
二、AFSCs向肝细胞诱导分化的体外实验研究
根据AFSCs的生物学特点,我们设计了三阶段AFSCs向肝细胞诱导分化的策略:二甲基亚砜(DMSO)及表皮生长因子预处理2天-序贯加入成纤维细胞生长因子4(FGF_4)、肝细胞生长因子(HGF)诱导分化10天-HGF、制瘤素(OSM)、地塞米松、ITS联合继续促进成熟10~+天。分别从细胞形态、肝细胞特定基因表达及肝细胞特殊功能检测等方面对诱导后细胞进行鉴定。
结果:诱导后AFSCs形态由梭形或长梭形逐渐向圆形或多角形转化,RT-PCR可检测到AFP、A1b、CK18、HNF4β、CYPB1等肝细胞特异基因的表达;免疫荧光法检测到胞浆内AFP及白蛋白的表达。诱导后的细胞能够摄取、排出靛青绿、贮存糖原、分泌白蛋白。表明AFSCs体外在一定条件下具有分化为有功能肝细胞的能力。
三、AFSCs向肝细胞诱导分化的体内研究
以四氯化碳(CCL_4)腹腔注射造成裸鼠急性肝损伤模型,尾静脉注入以荧光染料CFDA-SE标记的未诱导AFSCs。分别于移植后3天、10天、20天处死部分裸鼠,留取肝脏和血液标本,检测裸鼠肝脏内是否有标记细胞分布;同时观察细胞移植组及对照组肝脏病理及肝功能指标变化。
结果:移植后3天、10天、20天,裸鼠肝脏内均有一定比例的标记细胞,移植后10天标记细胞同时表达出成熟肝细胞标志-白蛋白,提示移植细胞已整合到裸鼠肝脏并分化为肝细胞;HE染色显示移植AFSCs的裸鼠肝脏损伤愈合过程加快,移植后10天组织学观察基本恢复正常;而对照组在移植后20天仍有明显的坏死和炎细胞浸润灶。
结论:本文成功建立了孕中期羊水来源干细胞分离培养及向肝细胞诱导分化的技术体系,表明羊水来源干细胞分离培养简单,体外扩增及分化能力强,经多种细胞因子三阶段诱导后,分化为具有肝细胞特征及功能的细胞,移植到肝损伤裸鼠模型后,能够整合到受体肝脏并向成熟肝细胞分化,并可促进宿主肝损伤的组织修复。
Amniotic fluid is a novel source of stem cells which has attracted intensive attentions in recent years.However,many questions remain uncertain about the methods of isolation and expansion of AFSCs,as well as their characteristics and differentiation potential.In this study,we conducted a set of experiments to acquire systematic knowledge about the properties of AFSCs;meanwhile,to evaluate whether AFSCs could be used for cell-based therapy,we induced them to differentiate into hepatic linage in vitro and in vivo.
Our study mainly included three parts of work.
1.Isolation,expansion,and biological analysis of human second-trimester AFSCs.
In order to compare the effect of different culture conditions on isolation and expansion of AFSCs,we primarily plated the amniotic fluid under 4 conditions: 10%of FBS(fetal bovine serum),20%of FBS,15%of FBS with 4ng/ml of bFGF, and 10%of FBS with gelatin- coating plate.The numbers of primary colonies and the capacity of expansion were compared.The stem cell-like characteristics analyzed included ultrastructure,growth kinetics,immunophenotypes,mRNA expressions,differentiation potential(to adipocyte,osteocyte and neuron) and karyotype analysis.
Results:we successfully obtained AFSCs under the condition of 15%of FBS with 4ng/ml of bFGF.AFSCs propagated persistently and rapidly.They shared surface markers of both mesenchymal and embryonic origins.After induced to adipocyte,osteocyte and neuron,they expressed specific markers of targeted cells revealed by RT-PCR or immunofluorescent staining.In addition,AFSCs were non-tumorigenic and kept normal karyotypes both at early and late passages.
2.Differentiation of AFSCs into hepatocyte in vitro.
This prompted us to design a three-stage strategy to induce AFSCs to differentiate into hepatic linage.The AFSCs were first pretreated with DMSO and EGF for 48 hours;then,they were differentiated into hepatic direction by sequential supplementation of FGF_4 and HGF for total 10 days;at last,they were matured under the functions of HGF,ITS,OSM and dexamethasone.
Our results showed that 90%of induced cells presented round-like or polygonal shape;AFP,Alb,CK18,HNF4β、CYPB1 genes were detected by RT-PCR or immunofluorescence.Furthermore,the induced AFSCs could intake and excrete ICG,store glycogen and secrete albumin,meaning that they acquired part of functions of matured hepatocyte.
3.Transplantation of AFSCs into CCL4-induced liver injury model of immunodefieient mice.
Acute liver injury of immunodeficient mice was induced by intraperitoneal administration of CCL_4.24 hours later,CFDA-SE labeled undifferentiated AFSCs were injected through tail vein.The mice were sacrificed after 3,10,and 20 days of transplantation and liver samples were examined.
Results:the labeled AFSCs were found in the mice liver after 3,10,20 days of transplantation under fluorescent microscope.Hepatocyte-differentiated AFSCs were identified by positive staining of human albumin after 10 days of transplantation.The liver of mice receiving AFSCs showed grossly normal histology after 10 days of transplantation;in contrast,pathological changes including infiltration of inflammatory cells and small foci of necrosis were still evident in that of sham-transplanted mice after 20 days
In summary,AFSCs could be isolated from second-trimester amniotic fluid under appropriate conditions.They harbor potent self-renewal and pluripotent capability while maintaining genetically stable.Their differentiation potential to hepatocyte in vitro and in vivo not only offered a novel method for treatment of liver disease,but also suggested that they might be a promising candidate for tissue engineering and stem cell therapy of other human disorders.
本研究主要包括三部分:
一、AFSCs分离培养方法的建立及生物学特性分析:
孕中期羊水(10-20ml)离心后细胞沉淀分别在下列四种不同条件下进行培养:低糖DMEM培养基(LD)+10%胎牛血清(FBS)、LD+20%胎牛血清、LD+15%胎牛血清+4ng/ml碱性成纤维细胞生长因子(bFGF)、LD+10%胎牛血清+0.1%明胶铺板。比较不同培养条件下原代集落数、可传代次数、扩增细胞数等;选择最佳条件对羊水来源干细胞进行扩增,通过形态学观察、细胞周期和生长曲线、特异性标志物检测、体外诱导分化、染色体核型分析及致瘤实验等对其生物学特性进行系统分析。
结果:15%血清结合bFGF可促进原代羊水细胞的贴壁生长和持续扩增,在此培养条件下AFSCs生长旺盛,持续传代;表达大部分间充质来源标志如CD105、CD44、CD29,以及部分全能干细胞标志如SSES-4、Oct-4和Nanog;经不同诱导分化条件培养后,从基因表达或表型上证实可向脂肪细胞、神经元及成骨细胞等不同胚层的目的细胞分化;长期培养中染色体核型稳定,注射裸鼠体内后8周未见肿瘤形成。
二、AFSCs向肝细胞诱导分化的体外实验研究
根据AFSCs的生物学特点,我们设计了三阶段AFSCs向肝细胞诱导分化的策略:二甲基亚砜(DMSO)及表皮生长因子预处理2天-序贯加入成纤维细胞生长因子4(FGF_4)、肝细胞生长因子(HGF)诱导分化10天-HGF、制瘤素(OSM)、地塞米松、ITS联合继续促进成熟10~+天。分别从细胞形态、肝细胞特定基因表达及肝细胞特殊功能检测等方面对诱导后细胞进行鉴定。
结果:诱导后AFSCs形态由梭形或长梭形逐渐向圆形或多角形转化,RT-PCR可检测到AFP、A1b、CK18、HNF4β、CYPB1等肝细胞特异基因的表达;免疫荧光法检测到胞浆内AFP及白蛋白的表达。诱导后的细胞能够摄取、排出靛青绿、贮存糖原、分泌白蛋白。表明AFSCs体外在一定条件下具有分化为有功能肝细胞的能力。
三、AFSCs向肝细胞诱导分化的体内研究
以四氯化碳(CCL_4)腹腔注射造成裸鼠急性肝损伤模型,尾静脉注入以荧光染料CFDA-SE标记的未诱导AFSCs。分别于移植后3天、10天、20天处死部分裸鼠,留取肝脏和血液标本,检测裸鼠肝脏内是否有标记细胞分布;同时观察细胞移植组及对照组肝脏病理及肝功能指标变化。
结果:移植后3天、10天、20天,裸鼠肝脏内均有一定比例的标记细胞,移植后10天标记细胞同时表达出成熟肝细胞标志-白蛋白,提示移植细胞已整合到裸鼠肝脏并分化为肝细胞;HE染色显示移植AFSCs的裸鼠肝脏损伤愈合过程加快,移植后10天组织学观察基本恢复正常;而对照组在移植后20天仍有明显的坏死和炎细胞浸润灶。
结论:本文成功建立了孕中期羊水来源干细胞分离培养及向肝细胞诱导分化的技术体系,表明羊水来源干细胞分离培养简单,体外扩增及分化能力强,经多种细胞因子三阶段诱导后,分化为具有肝细胞特征及功能的细胞,移植到肝损伤裸鼠模型后,能够整合到受体肝脏并向成熟肝细胞分化,并可促进宿主肝损伤的组织修复。
Amniotic fluid is a novel source of stem cells which has attracted intensive attentions in recent years.However,many questions remain uncertain about the methods of isolation and expansion of AFSCs,as well as their characteristics and differentiation potential.In this study,we conducted a set of experiments to acquire systematic knowledge about the properties of AFSCs;meanwhile,to evaluate whether AFSCs could be used for cell-based therapy,we induced them to differentiate into hepatic linage in vitro and in vivo.
Our study mainly included three parts of work.
1.Isolation,expansion,and biological analysis of human second-trimester AFSCs.
In order to compare the effect of different culture conditions on isolation and expansion of AFSCs,we primarily plated the amniotic fluid under 4 conditions: 10%of FBS(fetal bovine serum),20%of FBS,15%of FBS with 4ng/ml of bFGF, and 10%of FBS with gelatin- coating plate.The numbers of primary colonies and the capacity of expansion were compared.The stem cell-like characteristics analyzed included ultrastructure,growth kinetics,immunophenotypes,mRNA expressions,differentiation potential(to adipocyte,osteocyte and neuron) and karyotype analysis.
Results:we successfully obtained AFSCs under the condition of 15%of FBS with 4ng/ml of bFGF.AFSCs propagated persistently and rapidly.They shared surface markers of both mesenchymal and embryonic origins.After induced to adipocyte,osteocyte and neuron,they expressed specific markers of targeted cells revealed by RT-PCR or immunofluorescent staining.In addition,AFSCs were non-tumorigenic and kept normal karyotypes both at early and late passages.
2.Differentiation of AFSCs into hepatocyte in vitro.
This prompted us to design a three-stage strategy to induce AFSCs to differentiate into hepatic linage.The AFSCs were first pretreated with DMSO and EGF for 48 hours;then,they were differentiated into hepatic direction by sequential supplementation of FGF_4 and HGF for total 10 days;at last,they were matured under the functions of HGF,ITS,OSM and dexamethasone.
Our results showed that 90%of induced cells presented round-like or polygonal shape;AFP,Alb,CK18,HNF4β、CYPB1 genes were detected by RT-PCR or immunofluorescence.Furthermore,the induced AFSCs could intake and excrete ICG,store glycogen and secrete albumin,meaning that they acquired part of functions of matured hepatocyte.
3.Transplantation of AFSCs into CCL4-induced liver injury model of immunodefieient mice.
Acute liver injury of immunodeficient mice was induced by intraperitoneal administration of CCL_4.24 hours later,CFDA-SE labeled undifferentiated AFSCs were injected through tail vein.The mice were sacrificed after 3,10,and 20 days of transplantation and liver samples were examined.
Results:the labeled AFSCs were found in the mice liver after 3,10,20 days of transplantation under fluorescent microscope.Hepatocyte-differentiated AFSCs were identified by positive staining of human albumin after 10 days of transplantation.The liver of mice receiving AFSCs showed grossly normal histology after 10 days of transplantation;in contrast,pathological changes including infiltration of inflammatory cells and small foci of necrosis were still evident in that of sham-transplanted mice after 20 days
In summary,AFSCs could be isolated from second-trimester amniotic fluid under appropriate conditions.They harbor potent self-renewal and pluripotent capability while maintaining genetically stable.Their differentiation potential to hepatocyte in vitro and in vivo not only offered a novel method for treatment of liver disease,but also suggested that they might be a promising candidate for tissue engineering and stem cell therapy of other human disorders.
引文
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