人胚胎干細胞向胰島素分泌細胞誘導分化的研究
發(fā)布時間：2019-06-01 15:46
【摘要】： 第一章人胚胎干細胞(hESCs)向胰島素分泌細胞誘導分化的研究 目的:體外模擬胰腺的體內發(fā)育過程,通過添加因子的方式誘導hESCs得到胰島素分泌細胞。 方法:HESCs在人源性飼養(yǎng)層培養(yǎng)體系下傳代培養(yǎng),在傳代第5天開始誘導實驗,分四個階段。第一階段,hESCs在高濃度的ActivinA(100ng/ml)和低濃度血清條件下作用5天向限定性內胚層(DE)細胞特化;第二階段,先由視黃酸(10~(-5)M)處理24小時以啟動胰腺命運特化,第三階段,加入1%ITS和Fibronectin(5ug/ml)以及Exendin-4(50ng/ml)的作用7天,促進胰腺前體細胞的增殖和分化;第四階段,將細胞消化后懸浮培養(yǎng),加入1%N2,2%B27,煙堿(10~(-2)M),Exendin-4(50ng/ml)以及Betacellulin(20ng/ml)作用7天使細胞進一步分化成熟得到胰島樣細胞團。 結果:誘導過程中能檢測到胰腺發(fā)育關鍵轉錄因子(Sox17,Pdx1,Ngn3,Beta2)的表達,第5天有80%左右的Sox17陽性細胞,第13天有17%左右的Pdx1陽性細胞,第20天有4%左右的胰島素(Insulin)陽性細胞。誘導終末團塊表達胰島細胞標記蛋白Insulin和胰高血糖素(Glucagon);胰腺細胞的特征基因Insulin,Glucagon,生長抑素(Somastatin,SS),淀粉酶(Amylase);分泌功能相關的標記基因PC2,PC1/3,SUR1,GCK;胰島素釋放實驗顯示,終末細胞團對不同糖濃度(5.5mM和25mM)具有反應性,在高濃度條件下的胰島素釋放量是低濃度條件下的1.5—4倍。電鏡檢測到胞漿中存在大量分泌性顆粒。 結論:四階段誘導法誘導所得終末細胞團具備胰島細胞的基本特征。 第二章人孤雌胚胎干細胞(phESCs)和正常hESCs向胰島素分泌細胞誘導分化的比較研究 目的:探究phESCs能否向胰島素分泌細胞分化以及它與正常hESCs在分化過程中是否存在差異。 方法:以正常來源的hESCs(chHES8),孤雌來源的hESCs(chHES32,chHES69)共三株細胞為材料,在分化的d0,d5,d13,d20收集細胞,通過RT-PCR,realtime PCR以及免疫熒光染色來比較它們的胰腺發(fā)育標記蛋白(Sox17,Pdx1,Insulin)陽性率,胰腺發(fā)育標記基因和印記基因的表達水平,以及增殖能力上的差異。 結果:在誘導過程中,phESCs來源的細胞能表達胰腺發(fā)育各個階段的標記基因和蛋白,終末細胞團對不同葡萄糖濃度刺激呈現(xiàn)反應性。與正常hESCs來源的細胞相比,phESCs來源的細胞Sox17的基因和蛋白表達量與它的表達水平相近;phESCs來源的細胞Insulin蛋白表達水平是它的1/2左右;三株細胞的增殖能力沒有統(tǒng)計學差異。大部分印記基因呈現(xiàn)典型的父源性或母源性表達,只有IGF2/H19這對印記基因在2株phESCs的體外分化過程中出現(xiàn)了逐漸增強的表達,與正常hESCs表達水平接近。 結論:phESCs具有分化為胰島素分泌細胞的能力,正常hESCs較phESCs更容易向胰島素分泌細胞誘導分化。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
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