【摘要】： 第一章人胚胎干細(xì)胞(hESCs)向胰島素分泌細(xì)胞誘導(dǎo)分化的研究 目的:體外模擬胰腺的體內(nèi)發(fā)育過(guò)程,通過(guò)添加因子的方式誘導(dǎo)hESCs得到胰島素分泌細(xì)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(kāi)始誘導(dǎo)實(shí)驗(yàn),分四個(gè)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?xún)?nèi)胚層(DE)細(xì)胞特化;第二階段,先由視黃酸(10~(-5)M)處理24小時(shí)以啟動(dòng)胰腺命運(yùn)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(jìn)胰腺前體細(xì)胞的增殖和分化;第四階段,將細(xì)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(xì)胞進(jìn)一步分化成熟得到胰島樣細(xì)胞團(tuán)。 結(jié)果:誘導(dǎo)過(guò)程中能檢測(cè)到胰腺發(fā)育關(guān)鍵轉(zhuǎn)錄因子(Sox17,Pdx1,Ngn3,Beta2)的表達(dá),第5天有80%左右的Sox17陽(yáng)性細(xì)胞,第13天有17%左右的Pdx1陽(yáng)性細(xì)胞,第20天有4%左右的胰島素(Insulin)陽(yáng)性細(xì)胞。誘導(dǎo)終末團(tuán)塊表達(dá)胰島細(xì)胞標(biāo)記蛋白Insulin和胰高血糖素(Glucagon);胰腺細(xì)胞的特征基因Insulin,Glucagon,生長(zhǎng)抑素(Somastatin,SS),淀粉酶(Amylase);分泌功能相關(guān)的標(biāo)記基因PC2,PC1/3,SUR1,GCK;胰島素釋放實(shí)驗(yàn)顯示,終末細(xì)胞團(tuán)對(duì)不同糖濃度(5.5mM和25mM)具有反應(yīng)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(cè)到胞漿中存在大量分泌性顆粒。 結(jié)論:四階段誘導(dǎo)法誘導(dǎo)所得終末細(xì)胞團(tuán)具備胰島細(xì)胞的基本特征。 第二章人孤雌胚胎干細(xì)胞(phESCs)和正常hESCs向胰島素分泌細(xì)胞誘導(dǎo)分化的比較研究 目的:探究phESCs能否向胰島素分泌細(xì)胞分化以及它與正常hESCs在分化過(guò)程中是否存在差異。 方法:以正常來(lái)源的hESCs(chHES8),孤雌來(lái)源的hESCs(chHES32,chHES69)共三株細(xì)胞為材料,在分化的d0,d5,d13,d20收集細(xì)胞,通過(guò)RT-PCR,realtime PCR以及免疫熒光染色來(lái)比較它們的胰腺發(fā)育標(biāo)記蛋白(Sox17,Pdx1,Insulin)陽(yáng)性率,胰腺發(fā)育標(biāo)記基因和印記基因的表達(dá)水平,以及增殖能力上的差異。 結(jié)果:在誘導(dǎo)過(guò)程中,phESCs來(lái)源的細(xì)胞能表達(dá)胰腺發(fā)育各個(gè)階段的標(biāo)記基因和蛋白,終末細(xì)胞團(tuán)對(duì)不同葡萄糖濃度刺激呈現(xiàn)反應(yīng)性。與正常hESCs來(lái)源的細(xì)胞相比,phESCs來(lái)源的細(xì)胞Sox17的基因和蛋白表達(dá)量與它的表達(dá)水平相近;phESCs來(lái)源的細(xì)胞Insulin蛋白表達(dá)水平是它的1/2左右;三株細(xì)胞的增殖能力沒(méi)有統(tǒng)計(jì)學(xué)差異。大部分印記基因呈現(xiàn)典型的父源性或母源性表達(dá),只有IGF2/H19這對(duì)印記基因在2株phESCs的體外分化過(guò)程中出現(xiàn)了逐漸增強(qiáng)的表達(dá),與正常hESCs表達(dá)水平接近。 結(jié)論:phESCs具有分化為胰島素分泌細(xì)胞的能力,正常hESCs較phESCs更容易向胰島素分泌細(xì)胞誘導(dǎo)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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

【共引文獻(xiàn)】

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