本文關(guān)鍵詞：不同氧濃度下人孤雌胚胎干細胞基因組印跡的初步研究　出處：《廣州醫(yī)學院》2012年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 人孤雌胚胎干細胞 低氧 多能性 人孤雌胚胎干細胞 印跡基因 氧濃度

【摘要】：人類胚胎干細胞（human embryonic stem cells, hESC）來源于人囊胚內(nèi)細胞團，能夠在體外無限增殖并且具有向三胚層分化的潛能。因此，hESC在再生醫(yī)學、胚胎發(fā)育以及藥物開發(fā)等領(lǐng)域具有廣闊的應用前景。然而，受精卵來源的hESC不僅因涉及胚胎的毀壞而存在較大的倫理爭議，而且干細胞移植后的免疫排斥至今仍是hESC應用于臨床的一大障礙。而人孤雌胚胎干細胞（Humanparthenogenetic embryonic stem cell, hpESC）的建立不需要破環(huán)人類胚胎，且其MHC等位基因多是純合子，免疫源性低，可極大減少移植后產(chǎn)生的免疫排斥反應，在再生醫(yī)學及干細胞治療上有巨大的應用前景。然而，由于缺少父系基因，這種細胞的表觀遺傳穩(wěn)定性還不很明確。hpESC在體外培養(yǎng)及傳代中表觀遺傳的穩(wěn)定是其臨床應用的前提。本研究選擇了表觀遺傳學的一個重要組成部分——基因組印跡，利用全基因組表達譜芯片以及實時熒光定量PCR等方法對不同氧濃度下2株hpESC及1株受精卵來源的hESC的基因組印跡穩(wěn)定性進行初步研究，探討低氧及長期培養(yǎng)對人胚胎干細胞表觀遺傳穩(wěn)定性的影響，評估孤雌及受精卵來源hESC的臨床應用安全性。本研究表明，在5%氧濃度的培養(yǎng)條件下，2株hpESC具有正常hESC的生物學特性且核型正常，并且低氧通過HIF-2a的上調(diào)增強了hESC及hpESC中多能性基因的表達。本研究發(fā)現(xiàn)，不同的氧濃度會引起hESC和hpESC中印跡基因表達水平的改變，而不同的印跡基因?qū)ρ鯘舛鹊姆磻煌�，而HIF可能是低氧調(diào)節(jié)人胚胎干細胞表觀遺傳穩(wěn)定性的關(guān)鍵因子。但是，盡管在低氧的條件下，體外的長期培養(yǎng)仍然會影響hESC和hpESC印跡基因表達的穩(wěn)定性。因此，對比氧濃度，體外的長期培養(yǎng)是影響hESC和hpESC臨床應用安全性的首要因素。 第一部分 低氧條件下培養(yǎng)人孤雌胚胎干細胞及其生物學特性鑒定 【研究目的】 1.建立穩(wěn)定的低氧培養(yǎng)體系（5%O2），為后續(xù)研究開展奠定基礎。 2.在5%氧濃度下，對本實驗室現(xiàn)有的2株人孤雌胚胎干細胞(FY-phES-018和chHES-32)以及1株正常受精卵來源的人胚胎干細胞（FY-hES-7）進行培養(yǎng)并對其進行胚胎干細胞特性的鑒定，為后續(xù)實驗提供材料。 【材料方法】 1.采用三氣培養(yǎng)箱（5%O2/5%CO2/90%N2）建立5%氧濃度的低氧培養(yǎng)體系，以傳統(tǒng)的常氧培養(yǎng)體系（21%O2）作為對照。 2.將本實驗室前期建立的1株hpESC系(FY-phES-018)、1株hESC系（FY-hES-7）和來源于人類干細胞國家工程研究中心的另一株hpESC系(chHES-32)在5%氧濃度的條件下，進行體外培養(yǎng)傳代。進行干細胞特異性抗原SSEA-3, SSEA-4,TRA-1-60, TRA-1-81等染色鑒定以及體外分化能力檢測。 3.體外培養(yǎng)hpESC和hESC保持未分化狀態(tài)，收集早晚代次的的細胞，提取基因組總RNA和DNA。 4. RT-PCR檢測干細胞未分化分子標記基因的表達情況。 5.實時熒光定量PCR檢測多能性基因（NANOG和SOX2）及低氧誘導因子（HIF-1a和HIF-2a）在不同氧濃度下hESC和hpESC中的表達水平。 6.對3株細胞系進行核型分析并對2株hpESC系進行短串聯(lián)重復序列（STR）檢測。 【結(jié)果】 1.建立了穩(wěn)定的低氧培養(yǎng)體系。低氧培養(yǎng)的2株hpESC和1株hESC均具有典型hESC克隆的形態(tài)，表達干細胞特異性抗原SSEA-3, SSEA-4, TRA-1-60, TRA-1-81并且具有在體外自發(fā)分化形成擬胚體的能力且表達三個胚層的分化標志基因。 2.未分化分子標記基因NANOG, SOX2, OCT4, THY1和REX-1均在hESC和hpESC中均呈陽性表達。 3. NANOG、SOX2和HIF-2a在低氧組同株細胞中的表達量高于常氧組，其差異具有統(tǒng)計學（P0.05）；而HIF-1a在不同氧濃度組同株細胞中的表達量差異沒有統(tǒng)計學意義（P0.05）。 4.2株hpESC在低氧條件下都保持正常的核型：46，XX。細胞系之間的STR位點顯示不一樣，證明來自不同的胚胎。 【結(jié)論】 1.2株hpESC系及1株hESC系在5%氧濃度的環(huán)境中能夠維持人胚胎干細胞的生物學特性，具有不斷增殖及分化的能力。 2.低氧通過上調(diào)HIF-2a的表達增強了hESC及hpESC中多能性基因NANOG和SOX2的表達。由于低氧（5%O2）更接近胚胎在體內(nèi)發(fā)育的生理氧濃度，所以低氧的培養(yǎng)體系可能更有利于胚胎干細胞的生長，這也為后續(xù)關(guān)于低氧的研究提供了可靠的細胞材料。 第二部分 不同氧濃度下人孤雌胚胎干細胞印跡基因的表達狀態(tài) 【研究目的】 對不同氧濃度下未分化hpESC的印跡基因表達狀態(tài)進行動態(tài)觀察比較，初步探討低氧對hESC及hpESC表觀遺傳學穩(wěn)定性的影響，明確低氧培養(yǎng)是否有利于維持hpESC的印跡穩(wěn)定性，評價hpESC的臨床應用可行性，為人類胚胎干細胞表觀遺傳學的深入研究提供科學數(shù)據(jù)。 【材料方法】 1.利用建立的低氧培養(yǎng)體系與實驗室傳統(tǒng)的常氧培養(yǎng)體系平行培養(yǎng)2株hpESC和1株本實驗室前期建立的受精卵來源的hESC。 2.采用全基因組表達譜芯片測定不同氧濃度下2株未分化hpESC和1株hESC中的印跡基因表達狀態(tài)，篩選出不同氧濃度下相同代次的細胞中發(fā)生差異表達的印跡基因以及長期低氧條件下發(fā)生差異表達的印跡基因。比較不同氧濃度下及長期低氧條件下各株干細胞印跡基因表達的變化率。 3.實時熒光定量PCR驗證部分差異性表達印跡基因的表達水平。 4.對hpESC的印跡基因表達譜進行聚類分析，采用Gene Ontology(GO) analysis對來源于同一細胞系在不同氧濃度下培養(yǎng)的干細胞中差異表達的印跡基因進行基因功能分析。 5.比較差異表達印跡基因在不同hpESC系中變化的異同點。 【結(jié)果】 1.在長期低氧培養(yǎng)條件下，F(xiàn)Y-hES-7晚代細胞（P52）與相同條件下的早代細胞（p37）相比，共檢測出47個印跡基因，發(fā)現(xiàn)30個印跡基因發(fā)生差異性表達（2倍），其中16個表達上調(diào)，，14個表達下調(diào)。通過GO分析發(fā)現(xiàn)這些印跡基因富集于轉(zhuǎn)錄調(diào)控，各系統(tǒng)器官的發(fā)育以及調(diào)節(jié)生物代謝過程等。 2.在長期低氧培養(yǎng)條件下，F(xiàn)Y-phES-018晚代細胞（P52）與相同條件下的早代細胞（p37）相比，檢測獲得48個印跡基因，發(fā)生差異性表達（2倍）的印跡基因29個，其中17個表達上調(diào)，12個表達下調(diào)；chHES-32晚代細胞（p52）與相同條件下的早代細胞（p37）相比，檢測獲得印跡基因78個，其中差異性表達印跡基因61個，表達上調(diào)的基因34個，下調(diào)基因27個。通過對這些差異性表達的印跡基因進行GO分析發(fā)現(xiàn)，這些印跡基因富集于轉(zhuǎn)錄調(diào)控，各系統(tǒng)器官發(fā)育與形態(tài)以及調(diào)節(jié)生物代謝過程。 3.對比5%氧濃度下同一細胞系的晚代細胞，21%氧濃度下FY-hES-7晚代細胞中共檢測獲得59個印跡基因，發(fā)生差異性表達（2倍）的印跡基因37個，其中23個基因表達上調(diào)，14個基因表達下調(diào)。這些基因的GO分析顯示：這些印跡基因富集于RNA代謝過程的調(diào)節(jié)，胞外區(qū)域部分以及生長因子活性。 4.對比5%氧濃度下同一細胞系的晚代細胞，21%氧濃度下FY-phES-018晚代細胞中檢測獲得印跡基因53個，發(fā)生差異性表達（2倍）的印跡基因34個，其中13個基因表達上調(diào)，21個基因表達下調(diào)；21%氧濃度下chHES-32晚代細胞中檢測獲得75個印跡基因，發(fā)生差異性表達（2倍）的印跡基因44個，其中表達發(fā)生上調(diào)的基因23個，下調(diào)的基因21個。通過對這些基因進行GO分析發(fā)現(xiàn)這些印跡基因富集于各系統(tǒng)器官的形成與發(fā)育，轉(zhuǎn)錄及轉(zhuǎn)錄因子活性以及生物調(diào)節(jié)功能等。 5.不同氧濃度下各株細胞系之間印跡基因變化率的差異不具有統(tǒng)計學意義（P0.05），其中hpESC與hESC之間的印跡基因變化率不具有統(tǒng)計學意義（P0.05）；2株hpESC細胞之間的印跡基因變化率的差異無統(tǒng)計學意義(P0.05)。長期低氧培養(yǎng)的3株細胞系印跡基因變化率的差異沒有統(tǒng)計學意義（P0.05）；hpESC與hESC之間的印跡基因變化率不具有統(tǒng)計學意義（P0.05）；但是在長期低氧培養(yǎng)后，chHES-32細胞印跡基因的變化率高于比FY-phES-018細胞，2株hpESC細胞之間的印跡基因變化率的差異具有統(tǒng)計學意義(P0.05)。 6.部分印跡基因的實時熒光定量PCR驗證結(jié)果與基因芯片結(jié)果一致。 7.基因聚類分析結(jié)果顯示，在FY-phES-018和FY-hES-7細胞系中，5%氧濃度下早代和晚代細胞的印跡基因表達譜接近，而兩種氧濃度下的chHES-32晚代細胞的印跡基因表達譜較接近。 8.在長期低氧的條件下，不同的hpESC系中相同的印跡基因發(fā)生變化的趨勢不同，這些基因包括GATA3, CYP1B1, NKX6-2, BRUNOL4, SNURF, MYEOV2,KLF14, LMX1B和PEG3。在不同氧濃度下各hpESC系中發(fā)生差異性表達的印跡基因中一些基因的變化趨勢不一致，這些基因包括CSF2, FAM50B, BRUNOL4和FUCA1。 【結(jié)論】 1.不同的氧濃度會引起hESC和hpESC中印跡基因表達水平的改變，而不同的印跡基因?qū)ρ鯘舛鹊姆磻煌?2. HIF可能是低氧調(diào)節(jié)人胚胎干細胞表觀遺傳穩(wěn)定性的關(guān)鍵因子。 3.盡管在低氧的條件下，體外的長期培養(yǎng)仍然會影響hESC和hpESC印跡基因表達的穩(wěn)定性。因此，對比氧濃度，體外的長期培養(yǎng)是影響hESC和hpESC臨床應用安全性的首要因素。
[Abstract]:Human embryonic stem cells (human embryonic stem cells, hESC) derived from inner cell mass, and can have three differentiation potential in vitro proliferation. Therefore, hESC in regenerative medicine, it has broad application prospects of the embryonic development and drug development and other fields. However, the source of hESC not only because of the fertilized egg the destruction of embryos and ethics relates to the controversial, but a major obstacle to stem cell transplantation immune rejection is still the clinical application of hESC. The human parthenogenetic embryonic stem cells (Humanparthenogenetic embryonic stem cell, hpESC) the broken ring does not need to establish human embryos, and the MHC allele is homozygous. Low immunogenicity, can greatly reduce the immune rejection after transplantation produced, in regenerative medicine and stem cell therapy has great application prospect. However, due to the lack of paternal genes, this cell Epigenetic stability is not very clear.HpESC in vitro cultured and passaged in epigenetic stability is a prerequisite for its clinical application. This study chose epigenetics is an important part of genomic imprinting, were studied using whole genome microarray and quantitative real-time PCR method in different oxygen concentration under the stability of genomic imprinting of 2 hpESC strains and 1 strains derived from fertilized hESC, to investigate the effect of hypoxia and long-term culture of human embryonic stem cells influence the apparent genetic stability, assess the clinical safety of parthenogenetic eggs and origin of hESC. This study shows that the culture conditions of 5% oxygen concentration, 2 strains of hpESC with normal hESC biological characteristics and normal karyotype, and hypoxia through upregulation of HIF-2a enhanced the expression of genes hESC and hpESC can. The study found that the oxygen concentration will cause hESC And hpESC imprinted gene expression level changes, while the reaction of imprinted genes of different oxygen concentrations, and HIF may be the hypoxic regulation of human embryonic stem cells are a key factor on genetic stability. However, although under hypoxic conditions, long-term in vitro culture will still affect the stability of the expression of hESC and hpESC imprinted genes. Therefore, comparison of oxygen concentration, the long-term culture in vitro is a primary factor affecting hESC and hpESC clinical safety.
Part one
Culture of human parthenogenetic embryonic stem cells and identification of their biological characteristics under hypoxia
[purpose]
1. to establish a stable hypoxia culture system (5%O2), which lays the foundation for the follow-up research.
2. at 5% oxygen concentration, 2 human parthenogenetic embryonic stem cells (FY-phES-018 and chHES-32) and 1 normal fertilized eggs derived from human embryonic stem cells (FY-hES-7) were cultured, and their embryonic stem cell characteristics were identified, providing materials for subsequent experiments.
[material method]
1. the three gas incubator (5%O2/5%CO2/90%N2) was used to establish the hypoxia culture system of 5% oxygen concentration, and the traditional normal oxygen culture system (21%O2) was used as the control.
2. 1 strains of hpESC in the previous establishment (FY-phES-018), 1 strains of hESC (FY-hES-7) and from human stem cell engineering research center of the state of another strain hpESC (chHES-32) in 5% oxygen concentration conditions, were cultured in vitro. Stem cell specific antigen SSEA-3, SSEA-4 TRA-1-60, TRA-1-81, etc. were identified and in vitro differentiation assay.
3. the undifferentiated state of hpESC and hESC in vitro was maintained, and the cells of the early and late generations were collected, and the total genomic RNA and DNA. were extracted.
4. RT-PCR was used to detect the expression of undifferentiated molecular marker genes in stem cells.
5. the expression level of multipotent gene (NANOG and SOX2) and hypoxia inducible factor (HIF-1a and HIF-2a) in hESC and hpESC at different oxygen concentrations was detected by real time fluorescence quantitative PCR.
6. of 3 cell lines were karyotype and 2 hpESC lines were detected by short tandem repeat (STR).
[results]
1. to establish a stable culture system. Hypoxic hypoxia 2 hpESC strains and 1 hESC strains were cloned with typical hESC morphology, the expression of stem cell specific antigen SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 with spontaneous differentiation in vitro differentiation marker gene expression and the ability of the embryo to three germ layers.
2. undifferentiated molecular marker genes NANOG, SOX2, OCT4, THY1 and REX-1 were all positive in hESC and hpESC.
3. NANOG, the expression of SOX2 and HIF-2a in the same cell in hypoxia group was higher than in normoxic group, the difference was statistically (P0.05); and the expression of HIF-1a in different oxygen concentration group monoecious cells in the difference was not statistically significant (P0.05).
4.2 strains of hpESC maintain normal karyotype under hypoxic conditions: 46, the STR loci between the XX. cell lines are different and prove to come from different embryos.
[Conclusion]
1.2 hpESC lines and 1 hESC lines can maintain the biological characteristics of human embryonic stem cells in the 5% oxygen concentration environment, and have the ability to proliferate and differentiate continuously.
2. hypoxia enhanced the expression of gene NANOG and SOX2 in hpESC and hESC can up regulate the expression of HIF-2a. Due to hypoxia (5%O2) are closer to embryonic development in vivo in physiological oxygen concentration, so the hypoxia culture system may be more conducive to the growth of embryonic stem cells, which also continued after the study of hypoxia provides the reliable cell material.
The second part
Expression of imprinted gene in human parthenogenetic embryonic stem cells under different oxygen concentrations
[purpose]
To compare the expression state of dynamic observation of imprinted genes in undifferentiated hpESC under different oxygen concentration, preliminary study of hypoxia on hESC and hpESC influence the apparent genetic stability, clear whether hypoxia is conducive to maintaining the stability of imprinted hpESC, clinical application feasibility evaluation of hpESC, to provide scientific data for further research on human embryonic stem cells were apparent genetics.
[material method]
1., we established parallel cultivation of 2 hpESC and 1 strains of hESC. from fertilized eggs.
2. using the whole genome microarray was measured under different oxygen concentration of 2 strains of undifferentiated imprinted genes in 1 strains of hESC and hpESC expression, were screened with different oxygen concentration on imprinted gene expression differences occur under the same primary cells and long-term hypoxia imprinted genes differentially expressed under different oxygen concentration and comparison. Long term hypoxia conditions of the stem cells the expression of imprinted genes.
3. real time fluorescence quantitative PCR showed that the expression level of the imprinted gene was partially expressed.
4. cluster analysis was performed on the imprinted gene expression profiles of hpESC, and Gene Ontology (GO) analysis was used to analyze the differentially expressed imprinted genes from stem cells derived from the same cell line at different oxygen concentrations.
5. the differences and similarities between the differentially expressed imprinted genes in different hpESC lines were compared.
[results]
1. in the long-term hypoxia condition, FY-hES-7 cells (P52 cells) in late generations under the same conditions of early generation (P37), detected a total of 47 imprinted genes, found differential expression of 30 imprinted genes (2 times), of which 16 up-regulated and 14 downregulated by GO analysis. Found these imprinted genes enriched in transcriptional regulation, each organ development and regulate the biological metabolism process.
2. in the long-term hypoxia condition, FY-phES-018 cells (P52 cells) in late generations under the same conditions of early generation (P37) in detection of 48 imprinted genes, occurrence of differential expression of imprinted genes (2 times) 29, of which 17 were up-regulated and 12 down regulated expression of chHES-32 cells in later generation; (p52) cells under the same conditions of early generation (P37) in detection of imprinted gene 78, the expression of 61 imprinted genes, 34 up-regulated genes and 27 down regulated genes were found by GO analysis. The expression of imprinted genes on these differences, these imprinted genes enriched in transcription the system of regulation, metabolism and organ development form and regulation.
3. comparison of 5% oxygen concentration in the same cell line late generation cells, 21% oxygen concentration FY-hES-7 late generation cells detected 59 imprinted genes, occurrence of differential expression of imprinted genes (2 times) 37, of which 23 genes were up-regulated and 14 genes down regulated. These genes GO analysis showed that: the regulation of these imprinted genes enriched in RNA metabolism, extracellular region and growth factor activity.
4. comparison of 5% oxygen concentration in the same cell line late generation cells, 21% oxygen concentration FY-phES-018 late generation cells detected 53 imprinted genes, occurrence of differential expression of imprinted genes (2 times) 34, of which 13 genes were up-regulated and 21 genes down regulated; 21% oxygen concentration chHES-32 late generation cell detection in 75 imprinted genes, occurrence of differential expression of imprinted genes (2 times) 44, of which the expression of 23 up-regulated genes and 21 down regulated genes. Through GO analysis found that the formation and development of these imprinted genes enriched in the organs of these genes, transcription and transcription factor activity and the regulation of biological functions.
The difference between the 5. cell lines under different oxygen concentrations of imprinted gene change rate was not statistically significant (P0.05), the changes of imprinted genes between hpESC and hESC was not statistically significant (P0.05); the difference of imprinted gene changes between the 2 strains of hpESC cells was not statistically significant (P0.05 3). Strain differences in chronic hypoxic cultured cell lines of imprinted gene variation rate was not statistically significant (P0.05); imprinted gene changes between hpESC and hESC was not statistically significant (P0.05); but in the long term after hypoxia, the changes of chHES-32 cells was higher than that of imprinting genes than FY-phES-018 cells, there was statistically significant difference in imprinted gene changes among the 2 strains of hpESC cell rate (P0.05).
The results of real time fluorescence quantitative PCR verification in the 6. part of the imprinted gene were consistent with the results of the gene chip.
7. gene cluster analysis showed that in FY-phES-018 and FY-hES-7 cell lines, the expression profiles of imprinted genes in early generation and late generation cells at 5% oxygen concentration were close to each other, while the expression profiles of imprinted genes in chHES-32 late generation cells at two oxygen concentrations were close.
8. in chronic hypoxic conditions, different changes of imprinted genes of different hpESC in the trend, these genes including GATA3, CYP1B1, NKX6-2, BRUNOL4, SNURF, MYEOV2, KLF14, LMX1B and PEG3. under different oxygen concentrations of the hpESC lines in the difference of the change trend of some genetic imprinted gene inconsistency in these genes, including CSF2, FAM50B, BRUNOL4 and FUCA1.
[Conclusion]
1. different oxygen concentrations may cause changes in the level of imprinted gene expression in hESC and hpESC, while different imprinted genes have different responses to oxygen concentration.
2. HIF may be a key factor in the epigenetic stability of human embryonic stem cells with hypoxia.
3., in spite of hypoxia, long-term culture in vitro can still affect the stability of hESC and hpESC imprinted gene expression. Therefore, contrasting oxygen concentration and long-term culture in vitro is the primary factor affecting the safety of hESC and hpESC clinical application.

【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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相關(guān)期刊論文 前3條

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3 ;Derivation and characterization of human embryonic stem cell lines from the Chinese population[J];遺傳學報;2011年01期

本文編號：1418239