【作者】 李进；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 干细胞工程学， 2008， 硕士

【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^-5M)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^-2M),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。更多还原

【Abstract】 Chapter 1 Study on inducing human embryonic stem cells （hESCs） into insulin producing cellsObjective: TO get insulin producing cells （IPCs） from human embryonic stem cells by adding factors into the system in vitro, which based on simulating the development process of Pancreas in vivo.Methods: HESCs were cultured on human embryonic fibroblasts and started the experiment on the fifth day after last passage, which was divided into Four steps. Activin A （100ng/ml） and low dosage serum were used to generate definite endoderm （DE） from hESCs for five days in the first stage. Secondly, we treated the cells with retinoic acid (10^-5M) for 24 hours to start the pancreatic specialization .Thirdly, we added 1 % ITS, Fibronectin （5ug/ml） and Exendin-4 （50ng/ml） to the medium for one week for further differentiation to pancreas precursor cells. In the forth stage, pancreas precursor cells were cultured in suspension with medium containing 1 %N2, 1 %B27, Nicotinamide (10^-2M) ,Exendin-4 （50ng/ml） and Betacellulin （20ng/ml） for another week to get the insulin producing cells.Results: Expression of critical transcription factors （Sox17, Pdx1, Ngn3, Beta2） can be detected along with the inducing advancement and there is about 80% Sox17 positive cells on day 5, about 20% Pdx1 positive cells on day 13 and about 4% Insulin positive cells on day 20. The final clusters can express the islet protein markers insulin and glucagon; pancreas special expression genes such as insulin, glucagon, somatostatin and amylase; secretion function correlated markers such as PC2, PCI/3, SUR1, GCK. In the insulin release experiment, the secretion of insulin by these cells corresponded to the variations in glucose levels （5.5mM and 25mM）, the insulin content in high concentration was from 1.5 to 4 times than that in low concentration. In addition, the IPCs contained numerous secretory granules determined by electron microscopy.Conclusion: the IPCs we obtained from hESC cells by four-stage induce method have the basic characters of pancreatic island.Chapter 2 The comparion study between human parthenogenetic embryonic stem cells （phESCs） and normal hESCs in inducing them into insulin producing cellsObjective: To search for whether we can induce phES cells into insulin producing cells （IPCs） or not, and further to find out whether there are difference in differentiation process between hES cells and phES cells.Methods: We selected normal hESCs （chHES8） and phESCs （chHES32 , chHES69） as materials and collected the cells on d0, d5, d13, d20 in differentiation process. Some indices such as the positive percent of pancreas protein markers （Sox17, Pdx1, Insulin） , the expression of pancreas special genes, imprinting genes and proliferation ability were detected and analyzed by RT-PCR, realtime PCR and immunohistochemistry.Results: In induce process, the cells from phESCs can express the special gene and protein markers of Pancreas. The final cells corresponded to the variations in glucose levels. Compared to the cells from hESCs, the gene and protein expression level of Sox17 of cells from phESCs is similar with that of cells from hESCs; the protein expression level of insulin of cells from phESCs is 1/2 of that of cells from hESCs; there is no statistical difference in proliferation ability. The expression of most imprinting genes are in a classic way according with they are paternal gene or maternal gene, but the expression of paired genes IGF2 /H19 presents gradual enhancement way in the differentiation process and the expression level is near to normal hESCs.Conclusion: The phESCs can be induced into insulin producing cells, and the hESCs is easier to be induced into insulin producing cells compared to the phESCs.更多还原