不同氧浓度下人孤雌胚胎干细胞基因组印迹的初步研究

发布时间：2018-01-13 08:26

  本文关键词：不同氧浓度下人孤雌胚胎干细胞基因组印迹的初步研究　出处：《广州医学院》2012年硕士论文　论文类型：学位论文

  更多相关文章： 人孤雌胚胎干细胞 低氧 多能性 人孤雌胚胎干细胞 印迹基因 氧浓度

【摘要】：人类胚胎干细胞（human embryonic stem cells, hESC）来源于人囊胚内细胞团，能够在体外无限增殖并且具有向三胚层分化的潜能。因此，hESC在再生医学、胚胎发育以及药物开发等领域具有广阔的应用前景。然而，受精卵来源的hESC不仅因涉及胚胎的毁坏而存在较大的伦理争议，而且干细胞移植后的免疫排斥至今仍是hESC应用于临床的一大障碍。而人孤雌胚胎干细胞（Humanparthenogenetic embryonic stem cell, hpESC）的建立不需要破环人类胚胎，且其MHC等位基因多是纯合子，免疫源性低，可极大减少移植后产生的免疫排斥反应，在再生医学及干细胞治疗上有巨大的应用前景。然而，由于缺少父系基因，这种细胞的表观遗传稳定性还不很明确。hpESC在体外培养及传代中表观遗传的稳定是其临床应用的前提。本研究选择了表观遗传学的一个重要组成部分——基因组印迹，利用全基因组表达谱芯片以及实时荧光定量PCR等方法对不同氧浓度下2株hpESC及1株受精卵来源的hESC的基因组印迹稳定性进行初步研究，探讨低氧及长期培养对人胚胎干细胞表观遗传稳定性的影响，评估孤雌及受精卵来源hESC的临床应用安全性。本研究表明，在5%氧浓度的培养条件下，2株hpESC具有正常hESC的生物学特性且核型正常，并且低氧通过HIF-2a的上调增强了hESC及hpESC中多能性基因的表达。本研究发现，不同的氧浓度会引起hESC和hpESC中印迹基因表达水平的改变，而不同的印迹基因对氧浓度的反应不同，而HIF可能是低氧调节人胚胎干细胞表观遗传稳定性的关键因子。但是，尽管在低氧的条件下，体外的长期培养仍然会影响hESC和hpESC印迹基因表达的稳定性。因此，对比氧浓度，体外的长期培养是影响hESC和hpESC临床应用安全性的首要因素。 第一部分 低氧条件下培养人孤雌胚胎干细胞及其生物学特性鉴定 【研究目的】 1.建立稳定的低氧培养体系（5%O2），为后续研究开展奠定基础。 2.在5%氧浓度下，对本实验室现有的2株人孤雌胚胎干细胞(FY-phES-018和chHES-32)以及1株正常受精卵来源的人胚胎干细胞（FY-hES-7）进行培养并对其进行胚胎干细胞特性的鉴定，为后续实验提供材料。 【材料方法】 1.采用三气培养箱（5%O2/5%CO2/90%N2）建立5%氧浓度的低氧培养体系，以传统的常氧培养体系（21%O2）作为对照。 2.将本实验室前期建立的1株hpESC系(FY-phES-018)、1株hESC系（FY-hES-7）和来源于人类干细胞国家工程研究中心的另一株hpESC系(chHES-32)在5%氧浓度的条件下，进行体外培养传代。进行干细胞特异性抗原SSEA-3, SSEA-4,TRA-1-60, TRA-1-81等染色鉴定以及体外分化能力检测。 3.体外培养hpESC和hESC保持未分化状态，收集早晚代次的的细胞，提取基因组总RNA和DNA。 4. RT-PCR检测干细胞未分化分子标记基因的表达情况。 5.实时荧光定量PCR检测多能性基因（NANOG和SOX2）及低氧诱导因子（HIF-1a和HIF-2a）在不同氧浓度下hESC和hpESC中的表达水平。 6.对3株细胞系进行核型分析并对2株hpESC系进行短串联重复序列（STR）检测。 【结果】 1.建立了稳定的低氧培养体系。低氧培养的2株hpESC和1株hESC均具有典型hESC克隆的形态，表达干细胞特异性抗原SSEA-3, SSEA-4, TRA-1-60, TRA-1-81并且具有在体外自发分化形成拟胚体的能力且表达三个胚层的分化标志基因。 2.未分化分子标记基因NANOG, SOX2, OCT4, THY1和REX-1均在hESC和hpESC中均呈阳性表达。 3. NANOG、SOX2和HIF-2a在低氧组同株细胞中的表达量高于常氧组，其差异具有统计学（P0.05）；而HIF-1a在不同氧浓度组同株细胞中的表达量差异没有统计学意义（P0.05）。 4.2株hpESC在低氧条件下都保持正常的核型：46，XX。细胞系之间的STR位点显示不一样，证明来自不同的胚胎。 【结论】 1.2株hpESC系及1株hESC系在5%氧浓度的环境中能够维持人胚胎干细胞的生物学特性，具有不断增殖及分化的能力。 2.低氧通过上调HIF-2a的表达增强了hESC及hpESC中多能性基因NANOG和SOX2的表达。由于低氧（5%O2）更接近胚胎在体内发育的生理氧浓度，所以低氧的培养体系可能更有利于胚胎干细胞的生长，这也为后续关于低氧的研究提供了可靠的细胞材料。 第二部分 不同氧浓度下人孤雌胚胎干细胞印迹基因的表达状态 【研究目的】 对不同氧浓度下未分化hpESC的印迹基因表达状态进行动态观察比较，初步探讨低氧对hESC及hpESC表观遗传学稳定性的影响，明确低氧培养是否有利于维持hpESC的印迹稳定性，评价hpESC的临床应用可行性，为人类胚胎干细胞表观遗传学的深入研究提供科学数据。 【材料方法】 1.利用建立的低氧培养体系与实验室传统的常氧培养体系平行培养2株hpESC和1株本实验室前期建立的受精卵来源的hESC。 2.采用全基因组表达谱芯片测定不同氧浓度下2株未分化hpESC和1株hESC中的印迹基因表达状态，筛选出不同氧浓度下相同代次的细胞中发生差异表达的印迹基因以及长期低氧条件下发生差异表达的印迹基因。比较不同氧浓度下及长期低氧条件下各株干细胞印迹基因表达的变化率。 3.实时荧光定量PCR验证部分差异性表达印迹基因的表达水平。 4.对hpESC的印迹基因表达谱进行聚类分析，采用Gene Ontology(GO) analysis对来源于同一细胞系在不同氧浓度下培养的干细胞中差异表达的印迹基因进行基因功能分析。 5.比较差异表达印迹基因在不同hpESC系中变化的异同点。 【结果】 1.在长期低氧培养条件下，FY-hES-7晚代细胞（P52）与相同条件下的早代细胞（p37）相比，共检测出47个印迹基因，发现30个印迹基因发生差异性表达（2倍），其中16个表达上调，，14个表达下调。通过GO分析发现这些印迹基因富集于转录调控，各系统器官的发育以及调节生物代谢过程等。 2.在长期低氧培养条件下，FY-phES-018晚代细胞（P52）与相同条件下的早代细胞（p37）相比，检测获得48个印迹基因，发生差异性表达（2倍）的印迹基因29个，其中17个表达上调，12个表达下调；chHES-32晚代细胞（p52）与相同条件下的早代细胞（p37）相比，检测获得印迹基因78个，其中差异性表达印迹基因61个，表达上调的基因34个，下调基因27个。通过对这些差异性表达的印迹基因进行GO分析发现，这些印迹基因富集于转录调控，各系统器官发育与形态以及调节生物代谢过程。 3.对比5%氧浓度下同一细胞系的晚代细胞，21%氧浓度下FY-hES-7晚代细胞中共检测获得59个印迹基因，发生差异性表达（2倍）的印迹基因37个，其中23个基因表达上调，14个基因表达下调。这些基因的GO分析显示：这些印迹基因富集于RNA代谢过程的调节，胞外区域部分以及生长因子活性。 4.对比5%氧浓度下同一细胞系的晚代细胞，21%氧浓度下FY-phES-018晚代细胞中检测获得印迹基因53个，发生差异性表达（2倍）的印迹基因34个，其中13个基因表达上调，21个基因表达下调；21%氧浓度下chHES-32晚代细胞中检测获得75个印迹基因，发生差异性表达（2倍）的印迹基因44个，其中表达发生上调的基因23个，下调的基因21个。通过对这些基因进行GO分析发现这些印迹基因富集于各系统器官的形成与发育，转录及转录因子活性以及生物调节功能等。 5.不同氧浓度下各株细胞系之间印迹基因变化率的差异不具有统计学意义（P0.05），其中hpESC与hESC之间的印迹基因变化率不具有统计学意义（P0.05）；2株hpESC细胞之间的印迹基因变化率的差异无统计学意义(P0.05)。长期低氧培养的3株细胞系印迹基因变化率的差异没有统计学意义（P0.05）；hpESC与hESC之间的印迹基因变化率不具有统计学意义（P0.05）；但是在长期低氧培养后，chHES-32细胞印迹基因的变化率高于比FY-phES-018细胞，2株hpESC细胞之间的印迹基因变化率的差异具有统计学意义(P0.05)。 6.部分印迹基因的实时荧光定量PCR验证结果与基因芯片结果一致。 7.基因聚类分析结果显示，在FY-phES-018和FY-hES-7细胞系中，5%氧浓度下早代和晚代细胞的印迹基因表达谱接近，而两种氧浓度下的chHES-32晚代细胞的印迹基因表达谱较接近。 8.在长期低氧的条件下，不同的hpESC系中相同的印迹基因发生变化的趋势不同，这些基因包括GATA3, CYP1B1, NKX6-2, BRUNOL4, SNURF, MYEOV2,KLF14, LMX1B和PEG3。在不同氧浓度下各hpESC系中发生差异性表达的印迹基因中一些基因的变化趋势不一致，这些基因包括CSF2, FAM50B, BRUNOL4和FUCA1。 【结论】 1.不同的氧浓度会引起hESC和hpESC中印迹基因表达水平的改变，而不同的印迹基因对氧浓度的反应不同。 2. HIF可能是低氧调节人胚胎干细胞表观遗传稳定性的关键因子。 3.尽管在低氧的条件下，体外的长期培养仍然会影响hESC和hpESC印迹基因表达的稳定性。因此，对比氧浓度，体外的长期培养是影响hESC和hpESC临床应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.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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