hESCs在hEF支持下向内胚层诱导及正常与孤雌hESCs向胰岛样细胞诱导比较研究
发布时间：2018-09-11 10:16
【摘要】： 本研究室于2002年自主建立了人类胚胎干细胞(hESCs)系,至今已建立存有240余株hESCs系库。前期工作中成功利用人源性饲养层(hEF)体系下培养的hESCs诱导分化为限定性内胚层细胞和胰腺前体细胞,积累了诱导分化的经验。在此基础上,本课题组对hESCs向限定性内胚层细胞及胰岛素分泌细胞体外分化的条件进行了摸索和优化,首次发现hEF细胞在诱导分化过程中也发挥着辅助支持作用,对其机制进行了初步研究,显示hEF主要通过分泌可溶性因子激活ActivinA信号和Wnt信号通路发挥作用；模拟hEF细胞的辅助作用加入Wnt3a后发现Wnt3a在早期hESCs向内胚层诱导过程中发挥作用的窗口期是诱导第1天,成功建立无饲养层条件下hESCs向限定性内胚层细胞及胰岛素分泌细胞的诱导体系,在此基础上将自主建系的孤雌来源hESCs诱导分化为胰岛素分泌细胞,并对正常来源hESCs和孤雌来源hESCs在增殖能力与诱导效率等方面进行了比较,初步探讨了孤雌和正常来源hESCs向胰岛素分泌细胞体外诱导分化效率、增殖能力以及印记基因表达上的差异。本研究分为3个部分,主要研究方法和结果如下： 第一章hEF体系培养的hESCs向限定性内胚层的诱导分化 目的：1、研究在hEF细胞上培养的hESCs在高浓度ActivinA作用下向限定性内胚层分化过程中内胚层发育相关基因及蛋白表达的动态变化是否遵循体内发育模式；2、研究hEF细胞培养体系下诱导所得限定性内胚层细胞的体内外进一步分化能力； 方法：采用已有的ActivinA+FBS(AS)方法诱导hEF体系上培养的hESCs,收集O小时,6小时,12小时,1天,2天,3天,4天,5天的细胞,免疫荧光染色计数Brachyury和Sox17阳性率；real-timePCR检测原肠作用、内中外胚层、多能性标记的表达水平变化；将诱导5天所得细胞植入SCID鼠腿部肌肉,2月后对移植物进行切片HE染色,组织学分型各胚层来源比例；并将诱导5天细胞加入视黄酸,烟碱,肠促胰岛素类似物,重组人β-细胞调节素等因子,体外诱导内胚层细胞进一步分化为胰岛素分泌细胞；进行免疫荧光染色内分泌标记,PCR检测胰腺相关标记表达。 结果：PCR结果显示,采用AS法诱导hEF体系下的hESCs,在诱导第1天,原条标记GSC、Mixl1和内中胚层共同前体标记Brachyury表达明显上升,至第2天达高峰。内胚层标记Foxa2和Sox17在第2天开始上升,至第3天达高峰。染色计数结果显示Brachyury在诱导第2天达到高峰,阳性细胞率为43.7±16.6%；Sox17在诱导4天时达高峰81.7±5.4%；将诱导5天细胞移植入SCID小鼠体内后,移植物组织分型显示约75%的细胞为内胚层来源组织,外胚层来源组织比例低于1%。体外进一步诱导后,在诱导18天细胞表达胰腺前体标记pdx1, ngn3和beta2,在诱导25天细胞表达胰腺内外分泌及功能相关基因。细胞团染色显示外层为c肽和胰高血糖素双阳性细胞。 结论：在hEF体系上hESCs诱导分化为限定性内胚层细胞的过程符合体内内胚层发育规律,经过原条、内中胚层共同前体、内胚层3个阶段,获得了效率较高(约80%)的限定性内胚层细胞。该细胞具有在体内外进一步分化为更成熟内胚层组织细胞的能力。 第二章hEF细胞支持限定性内胚层分化的机制研究 目的：1、研究有饲养层及无饲养层培养体系上hESCs向内胚层细胞的诱导效率差异；2、研究hEF细胞是否分泌可溶性因子对限定性内胚层诱导产生影响；3、研究hEF细胞在限定性内胚层诱导过程中表达发育早期关键信号(TGFβ和Wnt信号)情况；4、摸索Wnt3a在限定性内胚层诱导中的作用时间窗,建立无饲养层体系下限定性内胚层诱导体系。 方法：对有饲养层体系(人饲养层H组和鼠饲养层M组)和无饲养层体系(FF组)上培养的hESCs进行AS法诱导,取诱导第5天细胞进行Soxl7染色计数和半定量PCR检测内胚层相关基因表达情况；在FF组基础上加入hEF条件培养基进行诱导(CM+A组),在诱导第5天染色计数Soxl7阳性细胞比率,PCR检测原条,内中外胚层标记的表达；取AS法诱导过程中不同时间点的hEF细胞,PCR检测胚胎早期发育关键信号TGF-β和Wnt信号通路中分泌型因子的基因表达；取FF组和CM+A组诱导1天细胞,western blot检测Wnt信号下游β-catenin的蛋白表达变化；在FF组加入25ng/ml Wnt3a作用1—4天,对不同培养时间点的细胞进行Brachyury, Sox 17染色计数,PCR检测原条,内中外胚层,胚外内胚层标记的表达。 结果：采用AS法诱导有饲养层体系下hESCs在诱导第5天均获得约80%的Sox17阳性细胞率(H组82.0±8.9%和M组78.7±3.4%),而无饲养层培养体系下Sox17阳性细胞率较低(FF组22.7±5.6%)；加入hEF细胞条件培养基后Sox17阳性率上升至63.2±13.4%(CM+A组),PCR检测结果显示在诱导过程中hEF细胞表达高强度ActivinA, Nodal在使用诱导培养基后6h—12h也出现了轻微的上调表达；同时hEF细胞还表达Wnt信号通路抑制剂Dkk1和Dkk3,并且Dkk3在进入诱导过程后6h起即出现了明显的下调,至12h,24h呈逐渐减弱的趋势；Western blot结果显示CM+A组较FF组在诱导第1天Wnt下游因子β-catenin的表达更强,PCR检测Wnt下游靶基因Brachyury和c-myc的表达在CM+A组也较FF组更强,以上结果提示饲养层细胞在内胚层诱导过程中一方面通过分泌ActivinA/Nodal等因子对内胚层诱导起正面促进作用,另一方面通过减弱对Wnt信号的抑制作用,反向活化Wnt信号,对内胚层诱导发挥辅助作用。进一步在CM+A体系加入Wnt3a后发现,Wnt3a与ActivinA共同作用1-4天,均能促进限定性内胚层的分化,共同作用1天Sox17阳性率上升至85.2±3.8%,是最佳的内胚层诱导体系。 结论：hEF通过表达高强度的Activin/Nodal信号,以及减弱对Wnt信号的抑制作用对限定性内胚层分化发挥的辅助作用。在无饲养层体系上,采用Wnt3a和ActivinA联合作用1天,再单独用ActivinA培养2天能最有效促进限定性内胚层的出现。 第三章孤雌与正常来源hESCs向胰岛素分泌细胞分化比较研究 目的：探讨孤雌来源hESCs在体外诱导因素调节下向胰岛素分泌细胞体外分化的能力,以及与正常hESCs在分化效率、增殖能力以及印记基因表达上的差异。 方法：以正常来源的hESCs (chHES137),孤雌来源的hESCs (chHES32,chHES69)共三株细胞为材料,采用第二章中Wnt3a和ActivinA共同作用1天组的内胚层诱导方法,结合视黄酸、烟碱、肠促胰岛素类似物、重组人p-细胞调节素等因子,分为内胚层、原始肠管、胰腺前体、内分泌前体、胰岛素分泌细胞5个阶段诱导。在分化的d0, d3, d6,d9, d12收集细胞,通过免疫荧光染色和real-time PCR来比较胰腺发育标记蛋白(Sox17, Pdx1)阳性率,胰腺发育标记基因和印记基因的表达水平,通过Ki67染色比较增殖能力上的差异。在分化终末阶段,收集细胞进行胰岛素染色、胰腺相关功能基因检测、胰岛素和C肽释放实验检测。 结果：诱导过程中3株hESCs的胰腺发育阶段标记基因的表达遵循胰腺发育的规律,在诱导d3出现内胚层标记表达高峰,诱导d6出现原始肠管标记高峰,诱导d9为胰腺前体标记表达高峰,通过定量PCR检测显示大部分发育标记基因中chHES137和chHES69的表达明显高于chHES32, Sox17和Pdx1染色计数结果也显示chHES137和chHES69的阳性细胞率明显高于chHES32。印记基因的表达遵循着印记基因表达规律,父源性基因(PEG3,IGF2)在正常来源hESCs的表达明显强于两株孤雌来源的hESCs10倍以上。母源性印记基因(GNAS,GRB10)在正常来源hESCs的表达则明显弱于两株孤雌来源的hESCs。chHES137和chHES69诱导终末细胞团均表达胰腺功能相关基因,对不同葡萄糖浓度刺激呈现反应性。3株细胞的增殖能力没有统计学差异。 结论：在体外诱导过程中,hESCs的分化遵循着胰腺体内发育的规律,孤雌来源hESCs具有分化为胰岛素分泌细胞的能力,诱导效率和增殖能力上与正常hESCs相比未见明显差异；孤雌和正常hESCs在诱导分化过程中,印记基因的表达基本符合表观遗传学规律,并具有一定的时间依赖性。
[Abstract]:The human embryonic stem cells (hESCs) line was established independently in 2002. Up to now, more than 240 strains of hESCs have been established. In the previous work, hESCs cultured in the human feeder layer (hEF) system were successfully used to induce differentiation into limited endodermal cells and pancreatic precursor cells, and the experience of inducing differentiation was accumulated. The conditions under which hESCs differentiate into restricted endodermal cells and insulin-secreting cells in vitro were explored and optimized. For the first time, it was found that hEF cells also play a supporting role in the process of inducing differentiation. The mechanism of hEF was preliminarily studied. The results showed that hEF activates ActivinA signal and Wnt signal mainly by secreting soluble factors. The window period of Wnt3a in the induction of early hESCs into endoderm was found to be the first day of induction. The induction system of hESCs into restricted endoderm cells and insulin-secreting cells without feeder layer was successfully established. On this basis, parthenogenetic lines were established independently. The differentiation of insulin-secreting cells induced by hESCs from normal and parthenogenetic sources was compared. The differentiation efficiency, proliferation ability and imprinted gene expression of insulin-secreting cells induced by parthenogenetic and normal hESCs were preliminarily discussed. In the 3 part, the main research methods and results are as follows:
Chapter 1 induction and differentiation of hESCs from hEF system to restrictive endoderm
AIM: 1. To investigate whether the dynamic changes of genes and protein expression related to endoderm development during the differentiation of hESCs into restricted endoderm induced by high concentration of ActivinA in hEF cells follow in vivo developmental pattern; 2. To study the further differentiation ability of restricted endoderm cells induced by hEF cell culture system in vitro and in vivo. Power;
Methods: Activan A + FBS (AS) was used to induce hESCs cultured in hEF system, and the cells were collected for O, 6, 12, 1, 2, 3, 4 and 5 days. The positive rates of Brachyury and Sox17 were counted by immunofluorescence staining. Cells were implanted into the leg muscles of SCID mice, and the grafts were stained with HE 2 months later to histologically classify the proportion of embryonic layers. After 5 days of induction, cells were added with retinoic acid, nicotine, insulin-stimulating analogues, recombinant human beta-cell regulator and other factors to induce further differentiation of endodermal cells into insulin-secreting cells in vitro. PCR was used to detect the expression of pancreatic related markers.
Results: PCR results showed that the expression of GSC, Mixl1 and Brachyury increased significantly on the first day of induction, and reached the peak on the second day. Foxa 2 and Sox17 increased on the second day and reached the peak on the third day. At day 4, the positive cell rate was 43.7 6550 Pdx1, Ngn3 and beta2 were labeled with progenitor, and the pancreatic endocrine and functional related genes were expressed on 25 days after induction.
CONCLUSION: The process of inducing differentiation of hESCs into restricted endodermal cells in hEF system conforms to the development rule of endoderm in vivo. After three stages, the limited endodermal cells with high efficiency (about 80%) were obtained. The cells have further differentiated into more mature endoderm tissue in vitro and in vivo. Cell capacity.
The second chapter is about the mechanism of hEF cells supporting the restriction of endoderm differentiation.
AIM: 1. To study the difference of inducing efficiency of hESCs to endoderm cells in feeder layer and no feeder layer culture systems; 2. To study whether soluble factors secreted by hEF cells affect restrictive endoderm induction; 3. To study the expression of key early developmental signals (TGF beta and Wnt signals) during restrictive endoderm induction by hEF cells. 4. To explore the time window of Wnt3a in restrictive endoderm induction and establish a restrictive endoderm induction system without feeder layer.
Methods: hESCs cultured in feeder layer system (human feeder layer H group and mouse feeder layer M group) and no feeder layer system (FF group) were induced by AS method. The cells on the 5th day of induction were counted by Soxl7 staining and semi-quantitative PCR to detect the expression of endoderm-related genes. The ratio of Soxl7 positive cells was counted by staining on the 5th day of induction, and the expression of endoderm, ectoderm and endoderm markers were detected by PCR; the expression of the key signal TGF-beta and Wnt in the early embryonic development of hEF cells at different time points during AS induction was detected by PCR; the expression of the secretory type factor genes in the key signal TGF-beta and Wnt signaling pathway was detected by Western blot; the cells in FF group and CM+A group The expression of beta-catenin in the downstream of Wnt signal was measured, and the cells in FF group were treated with 25ng/ml Wnt 3A for 1-4 days. Brachyury and Sox 17 staining were used to count the cells at different culture time points, and the expression of labels in the original, endoderm, ectoderm and ectoderm were detected by PCR.
Results: On the 5th day of induction, 80% of SOX17 positive cells were obtained in hESCs with feeder layer (82.0 The results showed that high-intensity Activin A was expressed in hEF cells during induction, and the expression of Nodal was slightly up-regulated from 6 h to 12 h after induction. At the same time, hEF cells also expressed Wnt signaling pathway inhibitors Dkk1 and Dkk3, and Dkk3 was down-regulated from 6 h to 12 h and gradually decreased at 24 h after induction. Western blot showed that the expression of downstream Wnt factor beta-catenin in CM+A group was stronger than that in FF group on the first day of induction, and the expression of downstream target genes Brachyury and c-myc in CM+A group was stronger than that in FF group by PCR. These results suggest that feeder layer cells secrete Activin A/Nodal and other factors to endoderm during the induction of endoderm. Layer induction plays a positive role in promoting endoderm induction. On the other hand, Wnt signal is reversely activated by weakening the inhibition of Wnt signal. Further, after adding Wnt 3a to CM+A system, it is found that Wnt 3a and ActivinA together for 1-4 days can promote the differentiation of the limited endoderm, and the positive rate of Sox17 increases to 1 day. 85.2 + 3.8% is the best endoderm induction system.
CONCLUSION: hEF plays an auxiliary role in limiting endoderm differentiation by expressing high-intensity Activin/Nodal signal and weakening the inhibition of Wnt signal. In feeder-free system, the combination of Wnt3a and Activin A for one day and Activin A alone for two days can most effectively promote the emergence of restricted endoderm.
The third chapter is about the differentiation between hESCs and insulin producing cells from parthenogenetic and normal sources.
AIM: To investigate the ability of parthenogenetic hESCs to differentiate into insulin-secreting cells in vitro under the regulation of inducing factors, and the difference between parthenogenetic hESCs and normal hESCs in differentiation efficiency, proliferation ability and imprinted gene expression.
METHODS: Three normal hESCs (chHES 137) and parthenogenetic hESCs (chHES 32, chHES 69) cells were used to induce endodermis in the first day group treated with Wnt 3a and Activan A in the second chapter. The cells were divided into endodermis and primitive intestine tube by combining retinoic acid, nicotine, insulin-stimulating analogue and recombinant human P-cell regulator. Pancreatic precursors, endocrine precursors and insulin-secreting cells were induced in 5 stages. The differentiated cells were collected at d0, d3, d6, D9 and d12. The positive rates of pancreatic development marker protein (Sox17, Pdx1), the expression levels of pancreatic development marker genes and imprinted genes were compared by immunofluorescence staining and real-time PCR. At the end of differentiation, the cells were collected for insulin staining, pancreatic related function gene detection, insulin and C-peptide release assay.
Results: The expression of marker genes in the pancreatic development stage of three hESCs strains followed the law of pancreatic development. Endodermal marker expression peaked in d3, primitive intestinal marker peaked in d6, and pancreatic precursor marker expression peaked in d9. Quantitative PCR analysis showed that chHES137 and CH were the most of the marker genes. The expression of HES69 was significantly higher than that of chHES32. The results of Sox17 and Pdx1 staining also showed that the positive rate of chHES137 and chHES69 was significantly higher than that of chHES32. The expression of imprinted gene followed the expression pattern of imprinted gene. The expression of paternal gene (PEG3, IGF2) in normal hESCs was significantly higher than that in parthenogenetic hESCs by 10 times. The expression of imprinted gene (GNAS, GRB10) in normal hESCs was significantly weaker than that in parthenogenetic hESCs. chHES137 and chHES69 induced terminal cell mass, which expressed pancreatic function-related genes, and showed no significant difference in the proliferative capacity of the three cells.
Conclusion: The differentiation of hESCs in vitro follows the development rule of pancreas in vivo. Parthenogenetic hESCs have the ability to differentiate into insulin-secreting cells, and there is no significant difference in induction efficiency and proliferation ability compared with normal hESCs. Epigenetic regulation is time dependent.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
本文编号：2236401
[Abstract]:The human embryonic stem cells (hESCs) line was established independently in 2002. Up to now, more than 240 strains of hESCs have been established. In the previous work, hESCs cultured in the human feeder layer (hEF) system were successfully used to induce differentiation into limited endodermal cells and pancreatic precursor cells, and the experience of inducing differentiation was accumulated. The conditions under which hESCs differentiate into restricted endodermal cells and insulin-secreting cells in vitro were explored and optimized. For the first time, it was found that hEF cells also play a supporting role in the process of inducing differentiation. The mechanism of hEF was preliminarily studied. The results showed that hEF activates ActivinA signal and Wnt signal mainly by secreting soluble factors. The window period of Wnt3a in the induction of early hESCs into endoderm was found to be the first day of induction. The induction system of hESCs into restricted endoderm cells and insulin-secreting cells without feeder layer was successfully established. On this basis, parthenogenetic lines were established independently. The differentiation of insulin-secreting cells induced by hESCs from normal and parthenogenetic sources was compared. The differentiation efficiency, proliferation ability and imprinted gene expression of insulin-secreting cells induced by parthenogenetic and normal hESCs were preliminarily discussed. In the 3 part, the main research methods and results are as follows:
Chapter 1 induction and differentiation of hESCs from hEF system to restrictive endoderm
AIM: 1. To investigate whether the dynamic changes of genes and protein expression related to endoderm development during the differentiation of hESCs into restricted endoderm induced by high concentration of ActivinA in hEF cells follow in vivo developmental pattern; 2. To study the further differentiation ability of restricted endoderm cells induced by hEF cell culture system in vitro and in vivo. Power;
Methods: Activan A + FBS (AS) was used to induce hESCs cultured in hEF system, and the cells were collected for O, 6, 12, 1, 2, 3, 4 and 5 days. The positive rates of Brachyury and Sox17 were counted by immunofluorescence staining. Cells were implanted into the leg muscles of SCID mice, and the grafts were stained with HE 2 months later to histologically classify the proportion of embryonic layers. After 5 days of induction, cells were added with retinoic acid, nicotine, insulin-stimulating analogues, recombinant human beta-cell regulator and other factors to induce further differentiation of endodermal cells into insulin-secreting cells in vitro. PCR was used to detect the expression of pancreatic related markers.
Results: PCR results showed that the expression of GSC, Mixl1 and Brachyury increased significantly on the first day of induction, and reached the peak on the second day. Foxa 2 and Sox17 increased on the second day and reached the peak on the third day. At day 4, the positive cell rate was 43.7 6550 Pdx1, Ngn3 and beta2 were labeled with progenitor, and the pancreatic endocrine and functional related genes were expressed on 25 days after induction.
CONCLUSION: The process of inducing differentiation of hESCs into restricted endodermal cells in hEF system conforms to the development rule of endoderm in vivo. After three stages, the limited endodermal cells with high efficiency (about 80%) were obtained. The cells have further differentiated into more mature endoderm tissue in vitro and in vivo. Cell capacity.
The second chapter is about the mechanism of hEF cells supporting the restriction of endoderm differentiation.
AIM: 1. To study the difference of inducing efficiency of hESCs to endoderm cells in feeder layer and no feeder layer culture systems; 2. To study whether soluble factors secreted by hEF cells affect restrictive endoderm induction; 3. To study the expression of key early developmental signals (TGF beta and Wnt signals) during restrictive endoderm induction by hEF cells. 4. To explore the time window of Wnt3a in restrictive endoderm induction and establish a restrictive endoderm induction system without feeder layer.
Methods: hESCs cultured in feeder layer system (human feeder layer H group and mouse feeder layer M group) and no feeder layer system (FF group) were induced by AS method. The cells on the 5th day of induction were counted by Soxl7 staining and semi-quantitative PCR to detect the expression of endoderm-related genes. The ratio of Soxl7 positive cells was counted by staining on the 5th day of induction, and the expression of endoderm, ectoderm and endoderm markers were detected by PCR; the expression of the key signal TGF-beta and Wnt in the early embryonic development of hEF cells at different time points during AS induction was detected by PCR; the expression of the secretory type factor genes in the key signal TGF-beta and Wnt signaling pathway was detected by Western blot; the cells in FF group and CM+A group The expression of beta-catenin in the downstream of Wnt signal was measured, and the cells in FF group were treated with 25ng/ml Wnt 3A for 1-4 days. Brachyury and Sox 17 staining were used to count the cells at different culture time points, and the expression of labels in the original, endoderm, ectoderm and ectoderm were detected by PCR.
Results: On the 5th day of induction, 80% of SOX17 positive cells were obtained in hESCs with feeder layer (82.0 The results showed that high-intensity Activin A was expressed in hEF cells during induction, and the expression of Nodal was slightly up-regulated from 6 h to 12 h after induction. At the same time, hEF cells also expressed Wnt signaling pathway inhibitors Dkk1 and Dkk3, and Dkk3 was down-regulated from 6 h to 12 h and gradually decreased at 24 h after induction. Western blot showed that the expression of downstream Wnt factor beta-catenin in CM+A group was stronger than that in FF group on the first day of induction, and the expression of downstream target genes Brachyury and c-myc in CM+A group was stronger than that in FF group by PCR. These results suggest that feeder layer cells secrete Activin A/Nodal and other factors to endoderm during the induction of endoderm. Layer induction plays a positive role in promoting endoderm induction. On the other hand, Wnt signal is reversely activated by weakening the inhibition of Wnt signal. Further, after adding Wnt 3a to CM+A system, it is found that Wnt 3a and ActivinA together for 1-4 days can promote the differentiation of the limited endoderm, and the positive rate of Sox17 increases to 1 day. 85.2 + 3.8% is the best endoderm induction system.
CONCLUSION: hEF plays an auxiliary role in limiting endoderm differentiation by expressing high-intensity Activin/Nodal signal and weakening the inhibition of Wnt signal. In feeder-free system, the combination of Wnt3a and Activin A for one day and Activin A alone for two days can most effectively promote the emergence of restricted endoderm.
The third chapter is about the differentiation between hESCs and insulin producing cells from parthenogenetic and normal sources.
AIM: To investigate the ability of parthenogenetic hESCs to differentiate into insulin-secreting cells in vitro under the regulation of inducing factors, and the difference between parthenogenetic hESCs and normal hESCs in differentiation efficiency, proliferation ability and imprinted gene expression.
METHODS: Three normal hESCs (chHES 137) and parthenogenetic hESCs (chHES 32, chHES 69) cells were used to induce endodermis in the first day group treated with Wnt 3a and Activan A in the second chapter. The cells were divided into endodermis and primitive intestine tube by combining retinoic acid, nicotine, insulin-stimulating analogue and recombinant human P-cell regulator. Pancreatic precursors, endocrine precursors and insulin-secreting cells were induced in 5 stages. The differentiated cells were collected at d0, d3, d6, D9 and d12. The positive rates of pancreatic development marker protein (Sox17, Pdx1), the expression levels of pancreatic development marker genes and imprinted genes were compared by immunofluorescence staining and real-time PCR. At the end of differentiation, the cells were collected for insulin staining, pancreatic related function gene detection, insulin and C-peptide release assay.
Results: The expression of marker genes in the pancreatic development stage of three hESCs strains followed the law of pancreatic development. Endodermal marker expression peaked in d3, primitive intestinal marker peaked in d6, and pancreatic precursor marker expression peaked in d9. Quantitative PCR analysis showed that chHES137 and CH were the most of the marker genes. The expression of HES69 was significantly higher than that of chHES32. The results of Sox17 and Pdx1 staining also showed that the positive rate of chHES137 and chHES69 was significantly higher than that of chHES32. The expression of imprinted gene followed the expression pattern of imprinted gene. The expression of paternal gene (PEG3, IGF2) in normal hESCs was significantly higher than that in parthenogenetic hESCs by 10 times. The expression of imprinted gene (GNAS, GRB10) in normal hESCs was significantly weaker than that in parthenogenetic hESCs. chHES137 and chHES69 induced terminal cell mass, which expressed pancreatic function-related genes, and showed no significant difference in the proliferative capacity of the three cells.
Conclusion: The differentiation of hESCs in vitro follows the development rule of pancreas in vivo. Parthenogenetic hESCs have the ability to differentiate into insulin-secreting cells, and there is no significant difference in induction efficiency and proliferation ability compared with normal hESCs. Epigenetic regulation is time dependent.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前2条
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2 陈林君;孤雌生殖的胚胎干细胞研究进展[J];中华男科学;2004年01期
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