羊水來源干細(xì)胞的生物學(xué)特性分析及其向肝細(xì)胞誘導(dǎo)分化的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-12-13 03:04
【摘要】： 羊水來源干細(xì)胞(amniotic fluid-derived stem cells,AFSCs)是近幾年來倍受關(guān)注的新成體干細(xì)胞類型,有關(guān)羊水干細(xì)胞的分離培養(yǎng)、生物學(xué)特性分析、體內(nèi)外分化潛能等仍有很多問題需深入探討。在其誘導(dǎo)分化方向和應(yīng)用領(lǐng)域方面,我們針對(duì)我國(guó)肝病的發(fā)病形式和目前肝臟再生修復(fù)治療的限制性因素,以分離、培養(yǎng)和鑒定羊水來源干細(xì)胞、并將其在體內(nèi)外誘導(dǎo)分化為肝細(xì)胞為目標(biāo),建立相應(yīng)的技術(shù)方法,為人類羊水來源干細(xì)胞的基礎(chǔ)研究和臨床應(yīng)用奠定基礎(chǔ)。 本研究主要包括三部分: 一、AFSCs分離培養(yǎng)方法的建立及生物學(xué)特性分析: 孕中期羊水(10-20ml)離心后細(xì)胞沉淀分別在下列四種不同條件下進(jìn)行培養(yǎng):低糖DMEM培養(yǎng)基(LD)+10%胎牛血清(FBS)、LD+20%胎牛血清、LD+15%胎牛血清+4ng/ml堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、LD+10%胎牛血清+0.1%明膠鋪板。比較不同培養(yǎng)條件下原代集落數(shù)、可傳代次數(shù)、擴(kuò)增細(xì)胞數(shù)等;選擇最佳條件對(duì)羊水來源干細(xì)胞進(jìn)行擴(kuò)增,通過形態(tài)學(xué)觀察、細(xì)胞周期和生長(zhǎng)曲線、特異性標(biāo)志物檢測(cè)、體外誘導(dǎo)分化、染色體核型分析及致瘤實(shí)驗(yàn)等對(duì)其生物學(xué)特性進(jìn)行系統(tǒng)分析。 結(jié)果:15%血清結(jié)合bFGF可促進(jìn)原代羊水細(xì)胞的貼壁生長(zhǎng)和持續(xù)擴(kuò)增,在此培養(yǎng)條件下AFSCs生長(zhǎng)旺盛,持續(xù)傳代;表達(dá)大部分間充質(zhì)來源標(biāo)志如CD105、CD44、CD29,以及部分全能干細(xì)胞標(biāo)志如SSES-4、Oct-4和Nanog;經(jīng)不同誘導(dǎo)分化條件培養(yǎng)后,從基因表達(dá)或表型上證實(shí)可向脂肪細(xì)胞、神經(jīng)元及成骨細(xì)胞等不同胚層的目的細(xì)胞分化;長(zhǎng)期培養(yǎng)中染色體核型穩(wěn)定,注射裸鼠體內(nèi)后8周未見腫瘤形成。 二、AFSCs向肝細(xì)胞誘導(dǎo)分化的體外實(shí)驗(yàn)研究 根據(jù)AFSCs的生物學(xué)特點(diǎn),我們?cè)O(shè)計(jì)了三階段AFSCs向肝細(xì)胞誘導(dǎo)分化的策略:二甲基亞砜(DMSO)及表皮生長(zhǎng)因子預(yù)處理2天-序貫加入成纖維細(xì)胞生長(zhǎng)因子4(FGF_4)、肝細(xì)胞生長(zhǎng)因子(HGF)誘導(dǎo)分化10天-HGF、制瘤素(OSM)、地塞米松、ITS聯(lián)合繼續(xù)促進(jìn)成熟10~+天。分別從細(xì)胞形態(tài)、肝細(xì)胞特定基因表達(dá)及肝細(xì)胞特殊功能檢測(cè)等方面對(duì)誘導(dǎo)后細(xì)胞進(jìn)行鑒定。 結(jié)果:誘導(dǎo)后AFSCs形態(tài)由梭形或長(zhǎng)梭形逐漸向圓形或多角形轉(zhuǎn)化,RT-PCR可檢測(cè)到AFP、A1b、CK18、HNF4β、CYPB1等肝細(xì)胞特異基因的表達(dá);免疫熒光法檢測(cè)到胞漿內(nèi)AFP及白蛋白的表達(dá)。誘導(dǎo)后的細(xì)胞能夠攝取、排出靛青綠、貯存糖原、分泌白蛋白。表明AFSCs體外在一定條件下具有分化為有功能肝細(xì)胞的能力。 三、AFSCs向肝細(xì)胞誘導(dǎo)分化的體內(nèi)研究 以四氯化碳(CCL_4)腹腔注射造成裸鼠急性肝損傷模型,尾靜脈注入以熒光染料CFDA-SE標(biāo)記的未誘導(dǎo)AFSCs。分別于移植后3天、10天、20天處死部分裸鼠,留取肝臟和血液標(biāo)本,檢測(cè)裸鼠肝臟內(nèi)是否有標(biāo)記細(xì)胞分布;同時(shí)觀察細(xì)胞移植組及對(duì)照組肝臟病理及肝功能指標(biāo)變化。 結(jié)果:移植后3天、10天、20天,裸鼠肝臟內(nèi)均有一定比例的標(biāo)記細(xì)胞,移植后10天標(biāo)記細(xì)胞同時(shí)表達(dá)出成熟肝細(xì)胞標(biāo)志-白蛋白,提示移植細(xì)胞已整合到裸鼠肝臟并分化為肝細(xì)胞;HE染色顯示移植AFSCs的裸鼠肝臟損傷愈合過程加快,移植后10天組織學(xué)觀察基本恢復(fù)正常;而對(duì)照組在移植后20天仍有明顯的壞死和炎細(xì)胞浸潤(rùn)灶。 結(jié)論:本文成功建立了孕中期羊水來源干細(xì)胞分離培養(yǎng)及向肝細(xì)胞誘導(dǎo)分化的技術(shù)體系,表明羊水來源干細(xì)胞分離培養(yǎng)簡(jiǎn)單,體外擴(kuò)增及分化能力強(qiáng),經(jīng)多種細(xì)胞因子三階段誘導(dǎo)后,分化為具有肝細(xì)胞特征及功能的細(xì)胞,移植到肝損傷裸鼠模型后,能夠整合到受體肝臟并向成熟肝細(xì)胞分化,并可促進(jìn)宿主肝損傷的組織修復(fù)。
[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,
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
本文編號(hào)：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,
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 胡斯樂;蒙古羊羊水源干細(xì)胞的分離鑒定及其定向成骨分化研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2011年
2 盧智娟;豬羊水干細(xì)胞生物學(xué)特性檢測(cè)及其特異性誘導(dǎo)分化研究[D];西北農(nóng)林科技大學(xué);2010年
，本文編號(hào)：2375760
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2375760.html
最近更新
教材專著
