人胚胎干细胞向胰岛素分泌细胞诱导分化的研究
发布时间：2019-06-01 15:46
【摘要】： 第一章人胚胎干细胞(hESCs)向胰岛素分泌细胞诱导分化的研究 目的:体外模拟胰腺的体内发育过程,通过添加因子的方式诱导hESCs得到胰岛素分泌细胞。 方法:HESCs在人源性饲养层培养体系下传代培养,在传代第5天开始诱导实验,分四个阶段。第一阶段,hESCs在高浓度的ActivinA(100ng/ml)和低浓度血清条件下作用5天向限定性内胚层(DE)细胞特化;第二阶段,先由视黄酸(10~(-5)M)处理24小时以启动胰腺命运特化,第三阶段,加入1%ITS和Fibronectin(5ug/ml)以及Exendin-4(50ng/ml)的作用7天,促进胰腺前体细胞的增殖和分化;第四阶段,将细胞消化后悬浮培养,加入1%N2,2%B27,烟碱(10~(-2)M),Exendin-4(50ng/ml)以及Betacellulin(20ng/ml)作用7天使细胞进一步分化成熟得到胰岛样细胞团。 结果:诱导过程中能检测到胰腺发育关键转录因子(Sox17,Pdx1,Ngn3,Beta2)的表达,第5天有80%左右的Sox17阳性细胞,第13天有17%左右的Pdx1阳性细胞,第20天有4%左右的胰岛素(Insulin)阳性细胞。诱导终末团块表达胰岛细胞标记蛋白Insulin和胰高血糖素(Glucagon);胰腺细胞的特征基因Insulin,Glucagon,生长抑素(Somastatin,SS),淀粉酶(Amylase);分泌功能相关的标记基因PC2,PC1/3,SUR1,GCK;胰岛素释放实验显示,终末细胞团对不同糖浓度(5.5mM和25mM)具有反应性,在高浓度条件下的胰岛素释放量是低浓度条件下的1.5—4倍。电镜检测到胞浆中存在大量分泌性颗粒。 结论:四阶段诱导法诱导所得终末细胞团具备胰岛细胞的基本特征。 第二章人孤雌胚胎干细胞(phESCs)和正常hESCs向胰岛素分泌细胞诱导分化的比较研究 目的:探究phESCs能否向胰岛素分泌细胞分化以及它与正常hESCs在分化过程中是否存在差异。 方法:以正常来源的hESCs(chHES8),孤雌来源的hESCs(chHES32,chHES69)共三株细胞为材料,在分化的d0,d5,d13,d20收集细胞,通过RT-PCR,realtime PCR以及免疫荧光染色来比较它们的胰腺发育标记蛋白(Sox17,Pdx1,Insulin)阳性率,胰腺发育标记基因和印记基因的表达水平,以及增殖能力上的差异。 结果:在诱导过程中,phESCs来源的细胞能表达胰腺发育各个阶段的标记基因和蛋白,终末细胞团对不同葡萄糖浓度刺激呈现反应性。与正常hESCs来源的细胞相比,phESCs来源的细胞Sox17的基因和蛋白表达量与它的表达水平相近;phESCs来源的细胞Insulin蛋白表达水平是它的1/2左右;三株细胞的增殖能力没有统计学差异。大部分印记基因呈现典型的父源性或母源性表达,只有IGF2/H19这对印记基因在2株phESCs的体外分化过程中出现了逐渐增强的表达,与正常hESCs表达水平接近。 结论:phESCs具有分化为胰岛素分泌细胞的能力,正常hESCs较phESCs更容易向胰岛素分泌细胞诱导分化。
[Abstract]:Study on the induction and differentiation of human embryonic stem cells (hESCs) from human embryonic stem cells (hESCs) to insulin-secreting cells Objective: To simulate the in vivo development of the pancreas in vitro and to induce the secretion of insulin through the induction of hESCs by the addition of factor. Cells. Methods: HESCs were subcultured under the culture system of human-derived feeder, and the induction experiment was started on the 5th day of passage. In the first stage, hESCs were activated at high concentrations of Activin A (100 ng/ ml) and at low concentrations of serum for 5 days to the non-limiting endoderm (DE) cells; in the second stage,24 hours were first treated with retinoic acid (10 to (-5) M) to initiate the formation of the pancreas, and in the third stage,1% of ITS and Fibronectin (5 ug/ ml) and Exendin-4 (50 ng/ ml) were added. 7 days to promote the proliferation and differentiation of the precursor cells of the pancreas, and the fourth stage, after the cells were digested, the cells were suspended and cultured, and 1% of N2,2% B27, nicotine (10 to (-2) M), Exendin-4 (50 ng/ ml) and Betacellin (20 ng/ ml) were added to further differentiate and mature to obtain the pancreas. The results showed that the expression of the key transcription factors (Sox17, Pdx1, Ngn3, Beta2) in the pancreas was detected in the induction process, and about 80% of the Sox17 positive cells in the fifth day and about 17% of the Pdx1 positive cells in the 13th day and about 4% of the insulin in the 20th day (Insu lin) positive cells. The terminal mass was induced to express the islet cell marker protein Inulin and Glucagon; the characteristic gene of the pancreatic cell, Inulin, Glucagon, Somatostatin, SS, and Amylase; the marker gene PC2, PC1/3, SUR1, GCK, and the pancreas associated with the secretion function; The island release experiments show that the terminal cell groups have a reactivity to different sugar concentrations (5.5 mM and 25 mM) and that the amount of insulin release under high concentration conditions is a low concentration condition 1.5-to-4-fold lower than the other. The electron microscope was used to detect the deposit in the cytoplasm. A large number of secretory granules. Conclusion: The terminal cell mass is induced by four-stage induction method. The basic characteristics of islet cells were found in the second chapter. The second chapter was isolated from human embryonic stem cells (phESCs) and normal hESCs to the pancreas. Objective: To investigate whether phESCs can differentiate into insulin-secreting cells and it is normal HESCs (chHES32, chHES69) and hESCs (chHES32, chHES69) were used as the material, and the cells were collected in the differentiated d0, d5, d13 and d20, and their pancreatic development marker proteins were compared by RT-PCR, realtime PCR and immunofluorescence staining (So). x17, Pdx1, Inulin positive rate, pancreatic development marker gene and The expression level of the imprinted gene, as well as the difference in the ability of proliferation, is that the cells derived from the phESCs can express the marker genes of various stages of the development of the pancreas during induction. The expression level of the gene and protein of the phESCs-derived cells, Sox17, was similar to that of the cells from the normal hESCs-derived cells, and the level of the expression of the cell Inulin protein from the phESCs-derived cells was There was no statistical difference in the proliferation ability of Sanzhu cells. Most of the imprinted genes exhibited typical paternal or maternal expression, and only IGF2/ H19 showed that the imprinting gene was in the process of in vitro differentiation of two phESCs. Conclusion: phESCs have the ability to differentiate into insulin-secreting cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2490387
[Abstract]:Study on the induction and differentiation of human embryonic stem cells (hESCs) from human embryonic stem cells (hESCs) to insulin-secreting cells Objective: To simulate the in vivo development of the pancreas in vitro and to induce the secretion of insulin through the induction of hESCs by the addition of factor. Cells. Methods: HESCs were subcultured under the culture system of human-derived feeder, and the induction experiment was started on the 5th day of passage. In the first stage, hESCs were activated at high concentrations of Activin A (100 ng/ ml) and at low concentrations of serum for 5 days to the non-limiting endoderm (DE) cells; in the second stage,24 hours were first treated with retinoic acid (10 to (-5) M) to initiate the formation of the pancreas, and in the third stage,1% of ITS and Fibronectin (5 ug/ ml) and Exendin-4 (50 ng/ ml) were added. 7 days to promote the proliferation and differentiation of the precursor cells of the pancreas, and the fourth stage, after the cells were digested, the cells were suspended and cultured, and 1% of N2,2% B27, nicotine (10 to (-2) M), Exendin-4 (50 ng/ ml) and Betacellin (20 ng/ ml) were added to further differentiate and mature to obtain the pancreas. The results showed that the expression of the key transcription factors (Sox17, Pdx1, Ngn3, Beta2) in the pancreas was detected in the induction process, and about 80% of the Sox17 positive cells in the fifth day and about 17% of the Pdx1 positive cells in the 13th day and about 4% of the insulin in the 20th day (Insu lin) positive cells. The terminal mass was induced to express the islet cell marker protein Inulin and Glucagon; the characteristic gene of the pancreatic cell, Inulin, Glucagon, Somatostatin, SS, and Amylase; the marker gene PC2, PC1/3, SUR1, GCK, and the pancreas associated with the secretion function; The island release experiments show that the terminal cell groups have a reactivity to different sugar concentrations (5.5 mM and 25 mM) and that the amount of insulin release under high concentration conditions is a low concentration condition 1.5-to-4-fold lower than the other. The electron microscope was used to detect the deposit in the cytoplasm. A large number of secretory granules. Conclusion: The terminal cell mass is induced by four-stage induction method. The basic characteristics of islet cells were found in the second chapter. The second chapter was isolated from human embryonic stem cells (phESCs) and normal hESCs to the pancreas. Objective: To investigate whether phESCs can differentiate into insulin-secreting cells and it is normal HESCs (chHES32, chHES69) and hESCs (chHES32, chHES69) were used as the material, and the cells were collected in the differentiated d0, d5, d13 and d20, and their pancreatic development marker proteins were compared by RT-PCR, realtime PCR and immunofluorescence staining (So). x17, Pdx1, Inulin positive rate, pancreatic development marker gene and The expression level of the imprinted gene, as well as the difference in the ability of proliferation, is that the cells derived from the phESCs can express the marker genes of various stages of the development of the pancreas during induction. The expression level of the gene and protein of the phESCs-derived cells, Sox17, was similar to that of the cells from the normal hESCs-derived cells, and the level of the expression of the cell Inulin protein from the phESCs-derived cells was There was no statistical difference in the proliferation ability of Sanzhu cells. Most of the imprinted genes exhibited typical paternal or maternal expression, and only IGF2/ H19 showed that the imprinting gene was in the process of in vitro differentiation of two phESCs. Conclusion: phESCs have the ability to differentiate into insulin-secreting cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
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