人源性上皮细胞的分离鉴定及其向胰岛素分泌细胞诱导的初步研究
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摘要
干细胞是一类具有无限的或者永生的自我更新能力的细胞,能够产生至少一种类型的,高度分化的子代细胞。按照发生学来源,干细胞可分为胚胎干细胞(embryonic stem cell,ESC)和成体干细胞(adultstem cell,ASC)。成体干细胞是指存在于一种已经分化组织中的未分化细胞,这种细胞能够自我更新并且能够特化形成组成该类型组织的细胞。成体干细胞存在于机体的各种组织器官中。在多数情况下,成体干细胞分化为与其组织来源一致的细胞,但是在某些情况下,成体干细胞的分化并不遵循这个规律,表现出很强的跨系或跨胚层分化潜能。
本课题组使用特定的分离培养方法,从人胎盘羊膜上分离得到羊膜上皮细胞(human Amniotic Epithelial Cells,hAECs),体外进行hAECs原代及传代培养,对体外培养的hAECs进行了一系列生物学鉴定,优化培养体系,建立了具有一定标本量的hAECs细胞库。在此基础上,对hAECs向胰岛素分泌细胞体外定向诱导分化的条件进行了摸索,成功诱导hAECs生成胰岛素分泌细胞,并初步探讨了hAECs向胰岛素分泌细胞定向诱导分化的机制。本研究分为两大部分,主要研究方法和结果如下:
第一章人羊膜上皮细胞(human Amniotic Epithelial Cells,hAECs)的分离及其干细胞生物学特征的鉴定
目的:分离人羊膜上皮细胞,建立人羊膜上皮细胞库,检测人羊膜上皮细胞的干细胞生物学特征。
方法:收集人羊膜标本,采用自定义的标准进行分级评定,分Ⅰ、Ⅱ、Ⅲ级(具体分级标准见论文第一部分);台盼蓝排斥实验检测分离得到的hAECs活细胞百分比,绘制生长曲线评价不同级别羊膜标本分离得到hAECs的细胞数量和细胞活力。对hAECs的干细胞生物学特征进行四个方面的检测:RT-PCR检测胚胎干细胞全能性基因表达,细胞免疫荧光检测胚胎干细胞表面标记,碱性磷酸酶(AKP)染色检测hAECs未分化性以及细胞核型普通G显带检测其培养过程中染色体的稳定性。
结果:Ⅰ、Ⅱ、Ⅲ级羊膜标本所能收获hAECs数量不同,以约1dm~2面积羊膜而言,Ⅰ级羊膜可收获约(11.253±2.3)×10~7个细胞;Ⅱ级羊膜标本可收获约(8±1.9)×10~7个细胞;Ⅲ级羊膜标本可收获的细胞数量不足10~5个细胞且这些细胞中的大部分都不能正常贴壁。台盼蓝排斥实验结果显示,Ⅰ级羊膜标本活细胞百分比约为89.7%±2.1%;Ⅱ级羊膜标本活细胞百分比约为77.81%±3.4%;Ⅲ级羊膜标本活细胞百分比约为21.6%±4.5%。生长曲线的评价结果显示,细胞的分裂增殖能力随羊膜标本质量的降低明显减退。建立了一个小型羊膜细胞库,冻存hAECs达近300管。
hAECs细胞表现SSEA-4强阳性,TRA-1-60和TRA-1-81弱阳性;AKP染色呈现部分阳性。连续传代三次的hAECs全能性相关基因的表达检测结果显示:oct4、TERF1基因在三代hAECs中的表达未见明显差别;THY、nanog基因的表达在三代中呈现逐渐加强的趋势;LEFTY在第二代hAECs中表达最强,在另外两代hAECs中的表达无明显差别;SOX2基因在三代中的表达呈现下降趋势。染色体G显带分析的结果显示,体外经连续传代培养达至少10代的hAECs仍保持了其染色体数目不变和染色体普通G显带核型正常。
结论:1.人羊膜上皮细胞适合建库,已建立了一个小型的羊膜细胞库。2.建立了一种行之有效的hAECs分离方法及适合hAECs体外增殖和扩增的培养体系。3.原代及体外连续传代两代之内的hAECs在细胞生长形态,全能性基因表达,细胞表面标记以及碱性磷酸酶染色等方面均无明显区别;4.体外连续传代培养的hAECs可以保持其遗传学稳定性。
第二章人羊膜上皮细胞(human Amniotic Epithelial Cells,hEACs)向胰岛素分泌细胞诱导分化的初步研究
目的:研究尼克酰氨联合细胞因子N2的培养体系对人羊膜上皮细胞向胰岛素分泌细胞定向诱导分化的影响。
方法:体外扩增一代的hAECs接种至琼脂粉铺皿的细胞培养皿中,采用如下诱导体系:1)LoW-DMEM+EGF;2)Low-DMEM+N2+尼克酰氨+EGF。RT-PCR检测诱导后细胞insulin,glucagon,pdx1,nkx6.1,ngn3等胰腺α,β细胞发育相关基因的表达;流式细胞术分析细胞表面标记PDX1的表达;细胞免疫荧光检测胰岛素分泌细胞表面insulin,nestin和glucagon标记的表达;葡萄糖刺激的胰岛素释放实验检测诱导后细胞对葡萄糖的应激性。为优化诱导体系,使用半定量RT-PCR检测不同诱导时间点的hAECs细胞胰腺相关基因的表达,如insulin,glucagon,pdx1,nkx6.1,ngn3等;RT-PCR检测细胞外黏附因子E-cadherin基因表达情况。
结果:诱导后的hAECs表达pdx1,nkx6.1,insulin,pax6,Isl1,ngn3等胰腺α,β细胞相关基因,弱表达glucagon基因。诱导第三天hAECs开始表达PDX1,表达率约为21.1%±3.4%;诱导14天后的hAECs细胞表面insulin标记表达可达13.64%±1.6%;hAECs经诱导后形成的类胰岛细胞团样细胞具有生物学功能,能对不同浓度的葡萄糖刺激产生反应,在16.5mM浓度葡萄糖刺激下,其胰岛素释放量平均可达59.43±6.0 vU/ml。
对不同诱导时间点的hAECs基因表达分析结果显示:insulin基因和glucagon基因具有不同的表达趋势。insulin基因表达在第一周的时候上调,随后下降;glucagon基因在诱导前3天未见表达,诱导第4天时可以检测到表达,第10天出现表达高峰,随后表达下调。pdx1,nkx6.1等这些β细胞分化和胰岛素转录的重要转录因子的表达趋势与insulin的表达趋势相一致;ngn3基因表达处于一个相对稳定状态。没经诱导的hAECs和诱导8天时的hAECs均表达E-cadherin基因;但诱导14天后的hAECs未见E-cadherin基因的表达。
结论:hAECs细胞可以在体外被定向诱导分化形成胰岛素分泌细胞。
Chapter 1 The Isolation and Identification of Human Amniotic Epithelial Cells
Objectives:We aimed to purify the hAECs isolated from human amnion and to identify the stem cell related characteristics of them so that we could establish a cell bank for further studies.
Methods:We defined human amnions as classⅠ、Ⅱ、Ⅲdepending on the transparence and cleanliness of the amnions.Trypan-blue staining was used for detecting the ratio of living cells.The growth curve was drawn for judging the characteristics of cells isolated from different placentas of different classes.RT-PCR,immunocytofluorescental analysis and AKP dyeing were used respectively for identifying the stem cell characteristics of hAECs.The cell samples of different passages were collected and then semi-quantity RT-PCR was performed to examine the pluripotency-related genes expression,such as Oct-4,nanog,TERF1, THY1,LEFTYA,and Rex-1.Immunocytofluorescental analysis was taken to test the expression of stem cell related markers,such as TRA-61~+, TRA-80~+,SSEA-1~+,SSEA-3~+ and SSEA-4~+.AKP dyeing was performed to detect the AKP positive cells.Chromosome Giemsa band analysis was undertaken to evaluate the genetic stability of hAECs cultured continuously in vitro for 10 passages.
Results:1.We have collected 62 amnion samples,and established a hAE cell bank with frozen cell to the amount of 300 tubes.2.Anmion of classⅠis the most abundant with a hAECs yield of(11.253+2.3)×10~7 cells/dm~2.3.Trypan-blue staining showed that the ratio of living cells was more than 75%.4.Almost all of the totipotent genes expressed in each passage of hAECs.THY1 gene expression up-regulated in P0,P1, P2 gradually;LEFTYA,Sox2,Oct-4 and TERF1 gene showed the same levels among 3 passages.5.Immunocytofluorescent analysis showed that most of hAECs were SSEA-4~+,part of them were positive weakly to TRA-1-60,TRA-1-81.6.AKP dyeing suggested that AKP~+ cells existed in P0,P1,P2 hAECs.7.Chromosome Giemsa band analysis showed the chromosomes maintained normal,even cultured for 10 passages.
Conclusions:1.We established a miniature hAE cell bank for further studies.2.There are no obvious biological characteristic differences among P0,P1,P2 hAECs.
Chapter 2 The Differentiation of Human Amniotic Epithelial Cells to Insulin-secreting Cells
Objectives:To investigate the induction differentiation from hAECs into insulin-secreting cells by using culture medium supplied with Nicotina-mide and cytokine N2.
Methods:P1 hAECs was seeded in cell dish coated with agar powder.RT-PCR was performed to testing the expression of pancreatic development-related genes such as insulin,glucagon,pdx1,nkx6.1 and ngn3.Flow cytometry was used to detect the PDX1~+ cell. Immunofluorescence was performed to testβ-cell specific marker Insulin. Glucose-stimulated insulin release experiment was taken to test the reaction ability of induced hAECs.Semi-quantity RT-PCR was used to evaluate the chronological gene expression changes during a 14-day inductive course,such as pdx1,ngn3,pax6,nkx6.1,insulin and glucagon. RT-PCR was performed to detect the expression of adhesion factor-related gene E-cadherin.
Results:1.RT-PCR results showed that pdx1,nkx6.1,insulin,pax6, Isl1,ngn3 were expressed in hAECs after induction.2.PDX1~+ cell number amounted to 21.1%±3.4%after 3 days induction.3.Insulin~+ cells number achieved 13.64%±1.6%after 2 weeks.4.The average insulin release of hAECs reached about 59.43±6.0 vU/ml.5.The gene chronological expression analysis showed that the expression of insulin gene was first up-regulated in the first week and subsequently down-regulated,while the glucagon gene expression showed down regulation after the crest-time at day 10.Expression tendency of pdx1, nkx6.1 gene was similar to that of insulin gene.ngn3 were steadyly expressed in all of the samples.6.E-cadherin gene expressed in hAECs induced for 8 days,but not expressed in hAECs induced for 14 days.
Conclusions:hAECs could be differentiated into insulin-secreting cells in vitro by using the induction system.
本课题组使用特定的分离培养方法,从人胎盘羊膜上分离得到羊膜上皮细胞(human Amniotic Epithelial Cells,hAECs),体外进行hAECs原代及传代培养,对体外培养的hAECs进行了一系列生物学鉴定,优化培养体系,建立了具有一定标本量的hAECs细胞库。在此基础上,对hAECs向胰岛素分泌细胞体外定向诱导分化的条件进行了摸索,成功诱导hAECs生成胰岛素分泌细胞,并初步探讨了hAECs向胰岛素分泌细胞定向诱导分化的机制。本研究分为两大部分,主要研究方法和结果如下:
第一章人羊膜上皮细胞(human Amniotic Epithelial Cells,hAECs)的分离及其干细胞生物学特征的鉴定
目的:分离人羊膜上皮细胞,建立人羊膜上皮细胞库,检测人羊膜上皮细胞的干细胞生物学特征。
方法:收集人羊膜标本,采用自定义的标准进行分级评定,分Ⅰ、Ⅱ、Ⅲ级(具体分级标准见论文第一部分);台盼蓝排斥实验检测分离得到的hAECs活细胞百分比,绘制生长曲线评价不同级别羊膜标本分离得到hAECs的细胞数量和细胞活力。对hAECs的干细胞生物学特征进行四个方面的检测:RT-PCR检测胚胎干细胞全能性基因表达,细胞免疫荧光检测胚胎干细胞表面标记,碱性磷酸酶(AKP)染色检测hAECs未分化性以及细胞核型普通G显带检测其培养过程中染色体的稳定性。
结果:Ⅰ、Ⅱ、Ⅲ级羊膜标本所能收获hAECs数量不同,以约1dm~2面积羊膜而言,Ⅰ级羊膜可收获约(11.253±2.3)×10~7个细胞;Ⅱ级羊膜标本可收获约(8±1.9)×10~7个细胞;Ⅲ级羊膜标本可收获的细胞数量不足10~5个细胞且这些细胞中的大部分都不能正常贴壁。台盼蓝排斥实验结果显示,Ⅰ级羊膜标本活细胞百分比约为89.7%±2.1%;Ⅱ级羊膜标本活细胞百分比约为77.81%±3.4%;Ⅲ级羊膜标本活细胞百分比约为21.6%±4.5%。生长曲线的评价结果显示,细胞的分裂增殖能力随羊膜标本质量的降低明显减退。建立了一个小型羊膜细胞库,冻存hAECs达近300管。
hAECs细胞表现SSEA-4强阳性,TRA-1-60和TRA-1-81弱阳性;AKP染色呈现部分阳性。连续传代三次的hAECs全能性相关基因的表达检测结果显示:oct4、TERF1基因在三代hAECs中的表达未见明显差别;THY、nanog基因的表达在三代中呈现逐渐加强的趋势;LEFTY在第二代hAECs中表达最强,在另外两代hAECs中的表达无明显差别;SOX2基因在三代中的表达呈现下降趋势。染色体G显带分析的结果显示,体外经连续传代培养达至少10代的hAECs仍保持了其染色体数目不变和染色体普通G显带核型正常。
结论:1.人羊膜上皮细胞适合建库,已建立了一个小型的羊膜细胞库。2.建立了一种行之有效的hAECs分离方法及适合hAECs体外增殖和扩增的培养体系。3.原代及体外连续传代两代之内的hAECs在细胞生长形态,全能性基因表达,细胞表面标记以及碱性磷酸酶染色等方面均无明显区别;4.体外连续传代培养的hAECs可以保持其遗传学稳定性。
第二章人羊膜上皮细胞(human Amniotic Epithelial Cells,hEACs)向胰岛素分泌细胞诱导分化的初步研究
目的:研究尼克酰氨联合细胞因子N2的培养体系对人羊膜上皮细胞向胰岛素分泌细胞定向诱导分化的影响。
方法:体外扩增一代的hAECs接种至琼脂粉铺皿的细胞培养皿中,采用如下诱导体系:1)LoW-DMEM+EGF;2)Low-DMEM+N2+尼克酰氨+EGF。RT-PCR检测诱导后细胞insulin,glucagon,pdx1,nkx6.1,ngn3等胰腺α,β细胞发育相关基因的表达;流式细胞术分析细胞表面标记PDX1的表达;细胞免疫荧光检测胰岛素分泌细胞表面insulin,nestin和glucagon标记的表达;葡萄糖刺激的胰岛素释放实验检测诱导后细胞对葡萄糖的应激性。为优化诱导体系,使用半定量RT-PCR检测不同诱导时间点的hAECs细胞胰腺相关基因的表达,如insulin,glucagon,pdx1,nkx6.1,ngn3等;RT-PCR检测细胞外黏附因子E-cadherin基因表达情况。
结果:诱导后的hAECs表达pdx1,nkx6.1,insulin,pax6,Isl1,ngn3等胰腺α,β细胞相关基因,弱表达glucagon基因。诱导第三天hAECs开始表达PDX1,表达率约为21.1%±3.4%;诱导14天后的hAECs细胞表面insulin标记表达可达13.64%±1.6%;hAECs经诱导后形成的类胰岛细胞团样细胞具有生物学功能,能对不同浓度的葡萄糖刺激产生反应,在16.5mM浓度葡萄糖刺激下,其胰岛素释放量平均可达59.43±6.0 vU/ml。
对不同诱导时间点的hAECs基因表达分析结果显示:insulin基因和glucagon基因具有不同的表达趋势。insulin基因表达在第一周的时候上调,随后下降;glucagon基因在诱导前3天未见表达,诱导第4天时可以检测到表达,第10天出现表达高峰,随后表达下调。pdx1,nkx6.1等这些β细胞分化和胰岛素转录的重要转录因子的表达趋势与insulin的表达趋势相一致;ngn3基因表达处于一个相对稳定状态。没经诱导的hAECs和诱导8天时的hAECs均表达E-cadherin基因;但诱导14天后的hAECs未见E-cadherin基因的表达。
结论:hAECs细胞可以在体外被定向诱导分化形成胰岛素分泌细胞。
Chapter 1 The Isolation and Identification of Human Amniotic Epithelial Cells
Objectives:We aimed to purify the hAECs isolated from human amnion and to identify the stem cell related characteristics of them so that we could establish a cell bank for further studies.
Methods:We defined human amnions as classⅠ、Ⅱ、Ⅲdepending on the transparence and cleanliness of the amnions.Trypan-blue staining was used for detecting the ratio of living cells.The growth curve was drawn for judging the characteristics of cells isolated from different placentas of different classes.RT-PCR,immunocytofluorescental analysis and AKP dyeing were used respectively for identifying the stem cell characteristics of hAECs.The cell samples of different passages were collected and then semi-quantity RT-PCR was performed to examine the pluripotency-related genes expression,such as Oct-4,nanog,TERF1, THY1,LEFTYA,and Rex-1.Immunocytofluorescental analysis was taken to test the expression of stem cell related markers,such as TRA-61~+, TRA-80~+,SSEA-1~+,SSEA-3~+ and SSEA-4~+.AKP dyeing was performed to detect the AKP positive cells.Chromosome Giemsa band analysis was undertaken to evaluate the genetic stability of hAECs cultured continuously in vitro for 10 passages.
Results:1.We have collected 62 amnion samples,and established a hAE cell bank with frozen cell to the amount of 300 tubes.2.Anmion of classⅠis the most abundant with a hAECs yield of(11.253+2.3)×10~7 cells/dm~2.3.Trypan-blue staining showed that the ratio of living cells was more than 75%.4.Almost all of the totipotent genes expressed in each passage of hAECs.THY1 gene expression up-regulated in P0,P1, P2 gradually;LEFTYA,Sox2,Oct-4 and TERF1 gene showed the same levels among 3 passages.5.Immunocytofluorescent analysis showed that most of hAECs were SSEA-4~+,part of them were positive weakly to TRA-1-60,TRA-1-81.6.AKP dyeing suggested that AKP~+ cells existed in P0,P1,P2 hAECs.7.Chromosome Giemsa band analysis showed the chromosomes maintained normal,even cultured for 10 passages.
Conclusions:1.We established a miniature hAE cell bank for further studies.2.There are no obvious biological characteristic differences among P0,P1,P2 hAECs.
Chapter 2 The Differentiation of Human Amniotic Epithelial Cells to Insulin-secreting Cells
Objectives:To investigate the induction differentiation from hAECs into insulin-secreting cells by using culture medium supplied with Nicotina-mide and cytokine N2.
Methods:P1 hAECs was seeded in cell dish coated with agar powder.RT-PCR was performed to testing the expression of pancreatic development-related genes such as insulin,glucagon,pdx1,nkx6.1 and ngn3.Flow cytometry was used to detect the PDX1~+ cell. Immunofluorescence was performed to testβ-cell specific marker Insulin. Glucose-stimulated insulin release experiment was taken to test the reaction ability of induced hAECs.Semi-quantity RT-PCR was used to evaluate the chronological gene expression changes during a 14-day inductive course,such as pdx1,ngn3,pax6,nkx6.1,insulin and glucagon. RT-PCR was performed to detect the expression of adhesion factor-related gene E-cadherin.
Results:1.RT-PCR results showed that pdx1,nkx6.1,insulin,pax6, Isl1,ngn3 were expressed in hAECs after induction.2.PDX1~+ cell number amounted to 21.1%±3.4%after 3 days induction.3.Insulin~+ cells number achieved 13.64%±1.6%after 2 weeks.4.The average insulin release of hAECs reached about 59.43±6.0 vU/ml.5.The gene chronological expression analysis showed that the expression of insulin gene was first up-regulated in the first week and subsequently down-regulated,while the glucagon gene expression showed down regulation after the crest-time at day 10.Expression tendency of pdx1, nkx6.1 gene was similar to that of insulin gene.ngn3 were steadyly expressed in all of the samples.6.E-cadherin gene expressed in hAECs induced for 8 days,but not expressed in hAECs induced for 14 days.
Conclusions:hAECs could be differentiated into insulin-secreting cells in vitro by using the induction system.
引文
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