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
发布时间：2018-12-13 03:04
【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。
[Abstract]:Amniotic fluid-derived stem cell (AFSCs) is a new type of stem cell in recent years. In terms of its induction and differentiation direction and application field, we aim at the pathogenesis of liver disease in our country and the restrictive factors of the current liver regeneration and repair treatment, to separate, culture and identify the amniotic fluid-derived stem cells, and to induce the differentiation into the hepatocyte as the target in vitro. and a corresponding technical method is established to lay a foundation for the basic research and clinical application of the human amniotic fluid source stem cells. This study mainly includes Three-part: the establishment of the method for the separation and culture of AFSCs Biological characteristics analysis: The cell pellet was cultured under four different conditions: low-sugar DMEM medium (LD) + 1 after centrifugation of the medium-term amniotic fluid (10-20ml). 0% fetal bovine serum (FBS), LD + 20% fetal bovine serum, LD + 15% fetal bovine serum + 4ng/ ml basic fibroblast growth factor (bFGF), LD + 10% fetal bovine The serum + 0.1% gelatin plank was plated. The primary colony number, the number of passable times and the number of expanded cells were compared under different culture conditions. The best condition was chosen to amplify the amniotic fluid-derived stem cells, and the cell cycle and the growth curve and the specific standard were observed by morphological observation, cell cycle and growth curve. in vitro induction and differentiation, chromosome karyotype analysis and tumorigenic experiment, etc. Results: 15% serum combined with bFGF can promote the adherent growth and continuous expansion of primary amniotic fluid cells. In this condition, AFSCs grow vigorously and continuously, and most of the mesenchymal origin markers such as CD105, CD44, CD29, and some of the universal stem cell markers such as SSE are expressed. S-4, Oct-4 and Nanog; after the culture of different induction and differentiation conditions, the target cells of different germ layers, such as the fat cells, the neurons and the osteoblast, can be confirmed from the gene expression or the phenotype; and the karyotype of the chromosomes in the long-term culture is stable, No tumor formation was seen in 8 weeks after the injection of the naked mouse. In vitro experimental study of the induced differentiation of AFSCs to hepatocytes, according to the biological characteristics of AFSCs, we designed the three-stage AFSCs strategy for inducing differentiation of the hepatocytes: the pretreatment of the two-stage AFSCs to the hepatocyte-induced differentiation: the pretreatment of the two-stage AFSCs and the epidermal growth factor 4 (FGF _ 4) of fibroblast growth factor 4 (FGF _ 4) and hepatocyte growth factor (HGF) induced by hepatocyte growth factor (HGF) for 10 days-HGF and OSM (), the combination of dexamethasone and ITS continued to promote the maturation of 10 to + days, respectively, from the cell morphology, the specific gene expression of the liver cells, The results showed that after induction, the shape of AFSCs was gradually changed from the shape of the shuttle or the long-shuttle to the circular or polygonal shape, and the expression of AFP, A1b, CK18, HNF4, and CYPB1 were detected by RT-PCR. The expression of AFP and albumin in the cytoplasm was detected by immunofluorescence. After the induction, the cells can be taken up, the indigo green is discharged, the glycogen is stored, and the albumin is secreted. AFSCs have differentiation into active power in vitro under certain conditions The ability of the hepatocytes to be enhanced. In vivo studies of the induction and differentiation of the liver cells with carbon tetrachloride (CCl _ 4) resulted in the acute toxicity of the naked mouse. Part of the nude mice were sacrificed at 3 days, 10 days and 20 days after transplantation, and the liver and blood samples were taken to test the liver of the nude mice. The changes of liver pathology and liver function in the cell transplantation group and the control group were observed. The results showed that there was a certain proportion of the marker cells in the liver of the nude mice for 3 days, 10 days, 20 days after the transplantation, and the marker cells were labeled 10 days after the transplantation. At the same time, the mature hepatocyte marker-albumin was expressed, indicating that the transplanted cells had been fully integrated into the liver of the nude mice and differentiated into the liver cells; the HE staining showed that the healing process of the liver injury of the bare mouse after the transplantation of AFSCs was accelerated, and after the transplantation, 1 0-day histological observation basically returned to normal; and the control group still has obvious necrosis and inflammatory cell infiltration after 20 days after transplantation. Conclusion: This paper successfully established the technique of separating and culturing the amniotic fluid-derived stem cells in the medium-term pregnancy and inducing differentiation to the hepatocytes. the system has the advantages of simple separation and culture of the amniotic fluid source stem cells, strong in vitro amplification and differentiation capability,
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329
本文编号：2375760
[Abstract]:Amniotic fluid-derived stem cell (AFSCs) is a new type of stem cell in recent years. In terms of its induction and differentiation direction and application field, we aim at the pathogenesis of liver disease in our country and the restrictive factors of the current liver regeneration and repair treatment, to separate, culture and identify the amniotic fluid-derived stem cells, and to induce the differentiation into the hepatocyte as the target in vitro. and a corresponding technical method is established to lay a foundation for the basic research and clinical application of the human amniotic fluid source stem cells. This study mainly includes Three-part: the establishment of the method for the separation and culture of AFSCs Biological characteristics analysis: The cell pellet was cultured under four different conditions: low-sugar DMEM medium (LD) + 1 after centrifugation of the medium-term amniotic fluid (10-20ml). 0% fetal bovine serum (FBS), LD + 20% fetal bovine serum, LD + 15% fetal bovine serum + 4ng/ ml basic fibroblast growth factor (bFGF), LD + 10% fetal bovine The serum + 0.1% gelatin plank was plated. The primary colony number, the number of passable times and the number of expanded cells were compared under different culture conditions. The best condition was chosen to amplify the amniotic fluid-derived stem cells, and the cell cycle and the growth curve and the specific standard were observed by morphological observation, cell cycle and growth curve. in vitro induction and differentiation, chromosome karyotype analysis and tumorigenic experiment, etc. Results: 15% serum combined with bFGF can promote the adherent growth and continuous expansion of primary amniotic fluid cells. In this condition, AFSCs grow vigorously and continuously, and most of the mesenchymal origin markers such as CD105, CD44, CD29, and some of the universal stem cell markers such as SSE are expressed. S-4, Oct-4 and Nanog; after the culture of different induction and differentiation conditions, the target cells of different germ layers, such as the fat cells, the neurons and the osteoblast, can be confirmed from the gene expression or the phenotype; and the karyotype of the chromosomes in the long-term culture is stable, No tumor formation was seen in 8 weeks after the injection of the naked mouse. In vitro experimental study of the induced differentiation of AFSCs to hepatocytes, according to the biological characteristics of AFSCs, we designed the three-stage AFSCs strategy for inducing differentiation of the hepatocytes: the pretreatment of the two-stage AFSCs to the hepatocyte-induced differentiation: the pretreatment of the two-stage AFSCs and the epidermal growth factor 4 (FGF _ 4) of fibroblast growth factor 4 (FGF _ 4) and hepatocyte growth factor (HGF) induced by hepatocyte growth factor (HGF) for 10 days-HGF and OSM (), the combination of dexamethasone and ITS continued to promote the maturation of 10 to + days, respectively, from the cell morphology, the specific gene expression of the liver cells, The results showed that after induction, the shape of AFSCs was gradually changed from the shape of the shuttle or the long-shuttle to the circular or polygonal shape, and the expression of AFP, A1b, CK18, HNF4, and CYPB1 were detected by RT-PCR. The expression of AFP and albumin in the cytoplasm was detected by immunofluorescence. After the induction, the cells can be taken up, the indigo green is discharged, the glycogen is stored, and the albumin is secreted. AFSCs have differentiation into active power in vitro under certain conditions The ability of the hepatocytes to be enhanced. In vivo studies of the induction and differentiation of the liver cells with carbon tetrachloride (CCl _ 4) resulted in the acute toxicity of the naked mouse. Part of the nude mice were sacrificed at 3 days, 10 days and 20 days after transplantation, and the liver and blood samples were taken to test the liver of the nude mice. The changes of liver pathology and liver function in the cell transplantation group and the control group were observed. The results showed that there was a certain proportion of the marker cells in the liver of the nude mice for 3 days, 10 days, 20 days after the transplantation, and the marker cells were labeled 10 days after the transplantation. At the same time, the mature hepatocyte marker-albumin was expressed, indicating that the transplanted cells had been fully integrated into the liver of the nude mice and differentiated into the liver cells; the HE staining showed that the healing process of the liver injury of the bare mouse after the transplantation of AFSCs was accelerated, and after the transplantation, 1 0-day histological observation basically returned to normal; and the control group still has obvious necrosis and inflammatory cell infiltration after 20 days after transplantation. Conclusion: This paper successfully established the technique of separating and culturing the amniotic fluid-derived stem cells in the medium-term pregnancy and inducing differentiation to the hepatocytes. the system has the advantages of simple separation and culture of the amniotic fluid source stem cells, strong in vitro amplification and differentiation capability,
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329
【引证文献】
相关硕士学位论文 前2条
1 胡斯乐;蒙古羊羊水源干细胞的分离鉴定及其定向成骨分化研究[D];内蒙古农业大学;2011年
2 卢智娟;猪羊水干细胞生物学特性检测及其特异性诱导分化研究[D];西北农林科技大学;2010年
，本文编号：2375760
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