hESCs在hEF支持下向內(nèi)胚層誘導(dǎo)及正常與孤雌hESCs向胰島樣細胞誘導(dǎo)比較研究
發(fā)布時間：2018-09-11 10:16
【摘要】： 本研究室于2002年自主建立了人類胚胎干細胞(hESCs)系,至今已建立存有240余株hESCs系庫。前期工作中成功利用人源性飼養(yǎng)層(hEF)體系下培養(yǎng)的hESCs誘導(dǎo)分化為限定性內(nèi)胚層細胞和胰腺前體細胞,積累了誘導(dǎo)分化的經(jīng)驗。在此基礎(chǔ)上,本課題組對hESCs向限定性內(nèi)胚層細胞及胰島素分泌細胞體外分化的條件進行了摸索和優(yōu)化,首次發(fā)現(xiàn)hEF細胞在誘導(dǎo)分化過程中也發(fā)揮著輔助支持作用,對其機制進行了初步研究,顯示hEF主要通過分泌可溶性因子激活A(yù)ctivinA信號和Wnt信號通路發(fā)揮作用；模擬hEF細胞的輔助作用加入Wnt3a后發(fā)現(xiàn)Wnt3a在早期hESCs向內(nèi)胚層誘導(dǎo)過程中發(fā)揮作用的窗口期是誘導(dǎo)第1天,成功建立無飼養(yǎng)層條件下hESCs向限定性內(nèi)胚層細胞及胰島素分泌細胞的誘導(dǎo)體系,在此基礎(chǔ)上將自主建系的孤雌來源hESCs誘導(dǎo)分化為胰島素分泌細胞,并對正常來源hESCs和孤雌來源hESCs在增殖能力與誘導(dǎo)效率等方面進行了比較,初步探討了孤雌和正常來源hESCs向胰島素分泌細胞體外誘導(dǎo)分化效率、增殖能力以及印記基因表達上的差異。本研究分為3個部分,主要研究方法和結(jié)果如下： 第一章hEF體系培養(yǎng)的hESCs向限定性內(nèi)胚層的誘導(dǎo)分化 目的：1、研究在hEF細胞上培養(yǎng)的hESCs在高濃度ActivinA作用下向限定性內(nèi)胚層分化過程中內(nèi)胚層發(fā)育相關(guān)基因及蛋白表達的動態(tài)變化是否遵循體內(nèi)發(fā)育模式；2、研究hEF細胞培養(yǎng)體系下誘導(dǎo)所得限定性內(nèi)胚層細胞的體內(nèi)外進一步分化能力； 方法：采用已有的ActivinA+FBS(AS)方法誘導(dǎo)hEF體系上培養(yǎng)的hESCs,收集O小時,6小時,12小時,1天,2天,3天,4天,5天的細胞,免疫熒光染色計數(shù)Brachyury和Sox17陽性率；real-timePCR檢測原腸作用、內(nèi)中外胚層、多能性標記的表達水平變化；將誘導(dǎo)5天所得細胞植入SCID鼠腿部肌肉,2月后對移植物進行切片HE染色,組織學(xué)分型各胚層來源比例；并將誘導(dǎo)5天細胞加入視黃酸,煙堿,腸促胰島素類似物,重組人β-細胞調(diào)節(jié)素等因子,體外誘導(dǎo)內(nèi)胚層細胞進一步分化為胰島素分泌細胞；進行免疫熒光染色內(nèi)分泌標記,PCR檢測胰腺相關(guān)標記表達。 結(jié)果：PCR結(jié)果顯示,采用AS法誘導(dǎo)hEF體系下的hESCs,在誘導(dǎo)第1天,原條標記GSC、Mixl1和內(nèi)中胚層共同前體標記Brachyury表達明顯上升,至第2天達高峰。內(nèi)胚層標記Foxa2和Sox17在第2天開始上升,至第3天達高峰。染色計數(shù)結(jié)果顯示Brachyury在誘導(dǎo)第2天達到高峰,陽性細胞率為43.7±16.6%；Sox17在誘導(dǎo)4天時達高峰81.7±5.4%；將誘導(dǎo)5天細胞移植入SCID小鼠體內(nèi)后,移植物組織分型顯示約75%的細胞為內(nèi)胚層來源組織,外胚層來源組織比例低于1%。體外進一步誘導(dǎo)后,在誘導(dǎo)18天細胞表達胰腺前體標記pdx1, ngn3和beta2,在誘導(dǎo)25天細胞表達胰腺內(nèi)外分泌及功能相關(guān)基因。細胞團染色顯示外層為c肽和胰高血糖素雙陽性細胞。 結(jié)論：在hEF體系上hESCs誘導(dǎo)分化為限定性內(nèi)胚層細胞的過程符合體內(nèi)內(nèi)胚層發(fā)育規(guī)律,經(jīng)過原條、內(nèi)中胚層共同前體、內(nèi)胚層3個階段,獲得了效率較高(約80%)的限定性內(nèi)胚層細胞。該細胞具有在體內(nèi)外進一步分化為更成熟內(nèi)胚層組織細胞的能力。 第二章hEF細胞支持限定性內(nèi)胚層分化的機制研究 目的：1、研究有飼養(yǎng)層及無飼養(yǎng)層培養(yǎng)體系上hESCs向內(nèi)胚層細胞的誘導(dǎo)效率差異；2、研究hEF細胞是否分泌可溶性因子對限定性內(nèi)胚層誘導(dǎo)產(chǎn)生影響；3、研究hEF細胞在限定性內(nèi)胚層誘導(dǎo)過程中表達發(fā)育早期關(guān)鍵信號(TGFβ和Wnt信號)情況；4、摸索Wnt3a在限定性內(nèi)胚層誘導(dǎo)中的作用時間窗,建立無飼養(yǎng)層體系下限定性內(nèi)胚層誘導(dǎo)體系。 方法：對有飼養(yǎng)層體系(人飼養(yǎng)層H組和鼠飼養(yǎng)層M組)和無飼養(yǎng)層體系(FF組)上培養(yǎng)的hESCs進行AS法誘導(dǎo),取誘導(dǎo)第5天細胞進行Soxl7染色計數(shù)和半定量PCR檢測內(nèi)胚層相關(guān)基因表達情況；在FF組基礎(chǔ)上加入hEF條件培養(yǎng)基進行誘導(dǎo)(CM+A組),在誘導(dǎo)第5天染色計數(shù)Soxl7陽性細胞比率,PCR檢測原條,內(nèi)中外胚層標記的表達；取AS法誘導(dǎo)過程中不同時間點的hEF細胞,PCR檢測胚胎早期發(fā)育關(guān)鍵信號TGF-β和Wnt信號通路中分泌型因子的基因表達；取FF組和CM+A組誘導(dǎo)1天細胞,western blot檢測Wnt信號下游β-catenin的蛋白表達變化；在FF組加入25ng/ml Wnt3a作用1—4天,對不同培養(yǎng)時間點的細胞進行Brachyury, Sox 17染色計數(shù),PCR檢測原條,內(nèi)中外胚層,胚外內(nèi)胚層標記的表達。 結(jié)果：采用AS法誘導(dǎo)有飼養(yǎng)層體系下hESCs在誘導(dǎo)第5天均獲得約80%的Sox17陽性細胞率(H組82.0±8.9%和M組78.7±3.4%),而無飼養(yǎng)層培養(yǎng)體系下Sox17陽性細胞率較低(FF組22.7±5.6%)；加入hEF細胞條件培養(yǎng)基后Sox17陽性率上升至63.2±13.4%(CM+A組),PCR檢測結(jié)果顯示在誘導(dǎo)過程中hEF細胞表達高強度ActivinA, Nodal在使用誘導(dǎo)培養(yǎng)基后6h—12h也出現(xiàn)了輕微的上調(diào)表達；同時hEF細胞還表達Wnt信號通路抑制劑Dkk1和Dkk3,并且Dkk3在進入誘導(dǎo)過程后6h起即出現(xiàn)了明顯的下調(diào),至12h,24h呈逐漸減弱的趨勢；Western blot結(jié)果顯示CM+A組較FF組在誘導(dǎo)第1天Wnt下游因子β-catenin的表達更強,PCR檢測Wnt下游靶基因Brachyury和c-myc的表達在CM+A組也較FF組更強,以上結(jié)果提示飼養(yǎng)層細胞在內(nèi)胚層誘導(dǎo)過程中一方面通過分泌ActivinA/Nodal等因子對內(nèi)胚層誘導(dǎo)起正面促進作用,另一方面通過減弱對Wnt信號的抑制作用,反向活化Wnt信號,對內(nèi)胚層誘導(dǎo)發(fā)揮輔助作用。進一步在CM+A體系加入Wnt3a后發(fā)現(xiàn),Wnt3a與ActivinA共同作用1-4天,均能促進限定性內(nèi)胚層的分化,共同作用1天Sox17陽性率上升至85.2±3.8%,是最佳的內(nèi)胚層誘導(dǎo)體系。 結(jié)論：hEF通過表達高強度的Activin/Nodal信號,以及減弱對Wnt信號的抑制作用對限定性內(nèi)胚層分化發(fā)揮的輔助作用。在無飼養(yǎng)層體系上,采用Wnt3a和ActivinA聯(lián)合作用1天,再單獨用ActivinA培養(yǎng)2天能最有效促進限定性內(nèi)胚層的出現(xiàn)。 第三章孤雌與正常來源hESCs向胰島素分泌細胞分化比較研究 目的：探討孤雌來源hESCs在體外誘導(dǎo)因素調(diào)節(jié)下向胰島素分泌細胞體外分化的能力,以及與正常hESCs在分化效率、增殖能力以及印記基因表達上的差異。 方法：以正常來源的hESCs (chHES137),孤雌來源的hESCs (chHES32,chHES69)共三株細胞為材料,采用第二章中Wnt3a和ActivinA共同作用1天組的內(nèi)胚層誘導(dǎo)方法,結(jié)合視黃酸、煙堿、腸促胰島素類似物、重組人p-細胞調(diào)節(jié)素等因子,分為內(nèi)胚層、原始腸管、胰腺前體、內(nèi)分泌前體、胰島素分泌細胞5個階段誘導(dǎo)。在分化的d0, d3, d6,d9, d12收集細胞,通過免疫熒光染色和real-time PCR來比較胰腺發(fā)育標記蛋白(Sox17, Pdx1)陽性率,胰腺發(fā)育標記基因和印記基因的表達水平,通過Ki67染色比較增殖能力上的差異。在分化終末階段,收集細胞進行胰島素染色、胰腺相關(guān)功能基因檢測、胰島素和C肽釋放實驗檢測。 結(jié)果：誘導(dǎo)過程中3株hESCs的胰腺發(fā)育階段標記基因的表達遵循胰腺發(fā)育的規(guī)律,在誘導(dǎo)d3出現(xiàn)內(nèi)胚層標記表達高峰,誘導(dǎo)d6出現(xiàn)原始腸管標記高峰,誘導(dǎo)d9為胰腺前體標記表達高峰,通過定量PCR檢測顯示大部分發(fā)育標記基因中chHES137和chHES69的表達明顯高于chHES32, Sox17和Pdx1染色計數(shù)結(jié)果也顯示chHES137和chHES69的陽性細胞率明顯高于chHES32。印記基因的表達遵循著印記基因表達規(guī)律,父源性基因(PEG3,IGF2)在正常來源hESCs的表達明顯強于兩株孤雌來源的hESCs10倍以上。母源性印記基因(GNAS,GRB10)在正常來源hESCs的表達則明顯弱于兩株孤雌來源的hESCs。chHES137和chHES69誘導(dǎo)終末細胞團均表達胰腺功能相關(guān)基因,對不同葡萄糖濃度刺激呈現(xiàn)反應(yīng)性。3株細胞的增殖能力沒有統(tǒng)計學(xué)差異。 結(jié)論：在體外誘導(dǎo)過程中,hESCs的分化遵循著胰腺體內(nèi)發(fā)育的規(guī)律,孤雌來源hESCs具有分化為胰島素分泌細胞的能力,誘導(dǎo)效率和增殖能力上與正常hESCs相比未見明顯差異；孤雌和正常hESCs在誘導(dǎo)分化過程中,印記基因的表達基本符合表觀遺傳學(xué)規(guī)律,并具有一定的時間依賴性。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329
本文編號：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329
【參考文獻】
相關(guān)期刊論文 前2條
1 ;Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro[J];Chinese Science Bulletin;2003年04期
2 陳林君;孤雌生殖的胚胎干細胞研究進展[J];中華男科學(xué);2004年01期
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