【作者】 王芳；

【导师】 孙莹璞；

【作者基本信息】 郑州大学 ， 妇产科学， 2010， 博士

【摘要】 多囊卵巢综合征(polycystic ovary syndrome, PCOS)是常见的育龄期妇女的疾病。临床表现为不孕、稀发排卵或无排卵、高雄激素血症等,约50%的PCOS病人伴有肥胖。其病因复杂,发病机理不明,无明确有效的治疗及预防措施。遗传因素在该病的发病中发挥重要作用。肥胖近年来受到关注,因脂肪组织在PCOS的发病和症状的维持中起到重要的作用。人胚胎干细胞(human embryonic stem cells, hESCs)理论上可定向分化为人体内所有类型的细胞。近年来该研究的一个领域是建立携带有先天或者“转基因”的遗传病hESCs系。目前国内外尚无有关PCOS源性hESCs建系成功的报道。PCOS源性hESCs稳定建系后,避免了动物模型的诸多弊端,可以直接进行PCOS患者发病机理的研究；并且可以利用hESCs的无限传代及多向分化能力,可以保持其染色体稳定性,不同批次定向分化后仍可保持其一致性,方便进行基因水平的操作,具有组间对照的可比性。在体外培养过程中,避免了多次重复取患者体液或组织等痛苦。目前虽已有文献报道将hESCs成功诱导分化为脂肪细胞,但诱导分化效率普遍偏低,成为脂肪细胞相关研究的主要障碍,故提高诱导分化效率非常必要。本研究利用PCOS患者的废弃胚胎建立hESCs系并分化为脂肪细胞,进一步检测其脂代谢的功能,并和正常来源的hESCs分化的脂肪细胞进行对比,根据基因芯片技术准确、全面、快速的特点,筛选出PCOS差异表达的基因,并着重对筛选出的基因进行研究,以期从基因水平阐明PCOS病人肥胖的发病机制。本文就上述热点和难点问题从以下四个方面进行了相关研究：第一部分在非PCOS来源的胚胎干细胞建系方法成熟的基础上,进一步建立了PCOS来源人胚胎干细胞系,并对其意义进行探讨。第二部分将人胚胎干细胞定向分化为脂肪细胞,在提高分化效率方面进行了研究。第三部分在相对高效的诱导胚胎干细胞分化为脂肪细胞的基础上,应用基因芯片技术对PCOS来源和非PCOS来源的胚胎干细胞分化的脂肪细胞进行差异基因的筛选。第四部分对筛出的高表达基因NROB2在蛋白水平进行验证,研究其在胚胎干细胞分化的脂肪细胞中的分布与表达。第一部分：PCOS来源人胚胎干细胞建系方法的探讨及意义目的：建立PCOS和非PCOS来源的hESCs系,对建系方法进行探讨,并分析影响建系成功的因素。方法：1.收集郑州大学第一附属医院生殖医学中心PCOS和非PCOS来源的体外受精-胚胎移植(in vitro fertilization embryo transfer, IVF-ET)和卵胞浆内单精子显微注射-胚胎移植(intracytoplasmic sperm injection, ICSI-ET)病人无移植及冷冻价值的新鲜及解冻的废弃胚胎,均经过病人知情同意后用于实验。将收集到的废弃及解冻胚胎,序贯培养到囊胚。2.分别采用免疫法、机械法、全胚接种3种不同的方法分离囊胚的内细胞团,将其种植于小鼠胚胎成纤维细胞、小儿包皮成纤维细胞和小鼠胚胎成纤维细胞和人包皮成纤维细胞按1：1混合的饲养层上,采用机械法或胶原酶法反复传代纯化,建立人胚胎干细胞系,并完成鉴定。结果：1.本实验共收集401名非PCOS患者的924枚新鲜废弃胚胎和17名PCOS患者的59个新鲜废弃胚胎,解冻25名非PCOS患者的83枚冷冻胚胎和4名PCOS患者的19枚冷冻胚胎,均序贯培养到囊胚,共获得86枚内细胞团(inner cell mass,ICM)。2.采用免疫法和机械法分离优质囊胚的ICM,种植与三种不同的饲养层上,混合饲养层可以较好的维持干细胞的生长,并得出混合饲养层细胞密度达到0.6×105/cm2即能较好的维持hESCs的生长状态。3.用机械法传代优点多,克隆生长快,形态佳,不易分化,故采用机械法传代,成功建立了17株hESCs系,其中非PCOS来源的12株,PCOS来源的5株,均完成鉴定工作。结论：1.培养系统的选择及其稳定性的维持对hESCs成功建系至关重要。2.混合饲养层的建系率显著高于单一饲养层,免疫法和机械法分离内细胞团的效率相当,机械法传代可以避免核型的变异。3. PCOS源性hESCs的建立,为研究人体发育早期PCOS的发病机理、筛选候选基因、基因修复及基因治疗等方面提供了一个良好的体外模型。第二部分：人胚胎干细胞定向分化为脂肪细胞及分化效率的研究目的：探讨罗格列酮和bFGF对人胚胎干细胞向脂肪细胞分化效率的影响,并比较PCOS来源与非PCOS来源胚胎干细胞成脂分化的能力的差异。方法：1.实验材料为第一部分PCOS患者及非PCOS患者来源的胚胎建立的人胚胎干细胞系,其中非PCOS来源的3株,PCOS来源的3株。2.采用不同浓度的罗格列酮加入基础诱导分化培养基中,分别为含0、1μmol/L、10μmol/L、100μmol/L的罗格列酮；采用不同浓度的bFGF加入基础诱导分化培养基中,分别含0、1 ng/mL、5 ng/mL、25 ng/mL的bFGF。摸索出罗格列酮和bFGF搭配的最佳浓度。3.根据在分化的不同阶段加入罗格列酮和bFGF,进行诱导方案分组。4.采用油红0染色、甘油三酯测定、MTT比色法及RT-PCR法检测PRARy-2的表达得出脂肪细胞最多的诱导分化方案,5.在摸索出最佳的诱导方案和罗格列酮、bFGF的浓度后,比较PCOS来源与非PCOS来源：hESCs定向诱导分化为脂肪细胞能力的差异。结果：1.随着分化时间的延长,诱导分化第2周时,视野中可见散在成熟脂肪细胞,细胞内出现脂滴,小脂滴聚集并出现脂滴融合现象,细胞逐渐变为圆形或椭圆形,将胞核推向细胞一侧,hESCs分化的脂肪细胞内的脂滴被油红O染色特异性染为红色。2.根据分化后脂肪细胞中甘油三酯的含量可以估算脂肪细胞的分化效率,10μmol/L罗格列酮和5ng/mL bFGF诱导分化效率均显著高于其余3组(P<0.05),在诱导的后半周期加入上述两种诱导分化因子可以得到较多的脂肪细胞。3.PCOS来源hESCs定向诱导分化为脂肪细胞能力较非PCOS组强,但是无显著统计学差异结论：1.建立hESCs向脂肪细胞体外定向分化效率较高的实验模型,即10μmol/L罗格列酮和5ng/mL bFGF联合后半周期诱导得到的脂肪细胞最多。2.分化得到的脂肪细胞不仅具有脂肪细胞的形态,还就有聚集甘油三酯的功能,并具有成熟脂肪细胞的功能,为细胞水平研究脂肪细胞的分化和代谢提供了材料。第三部分：基因芯片对PCOS来源胚胎干细胞分化的脂肪细胞差异基因的筛选目的：以脂肪细胞作为研究工具,在mRNA水平上筛选出了一些PCOS相关的分子标志物及基因,从而为探索PCOS发病的分子机制提供线索和依据,为临床诊断与治疗提供了新的途径和可能靶标。方法：1.实验材料为3株PCOS来源的hESCs系(p-hESC-1, p-hESC-2, p-hESC-3)定向分化的脂肪细胞作为实验组1、2、3；1株非PCOS来源的hESCs系(ZZU-hESC-1))定向分化的脂肪细胞作为对照组。2.应用北京博奥晶芯(?)人类全基因组寡核苷酸微阵列芯片,采用基因芯片检测与分析PCOS来源的hESCs系定向分化的脂肪细胞和非PCOS来源的hESCs系定向分化的脂肪细胞的差异基因的比较。3．对筛选出的差异基因进行Real-time PCR验证结果：1.在芯片的分析结果中,共获得PCOS和非PCOS来源hESCs分化的脂肪细胞差异表达基因153个,其中上调表达91个,下调表达62个2.差异基因分别属于多个分子功能家庭,包括DNA结合、金属离子结合、水解酶、受体激活和转移酶等等；参与多个生物过程,包括糖代谢、脂代谢、细胞信号传递及调控等等,该部分差异基因占总数的一半；还有一部分差异基因位于不同的细胞组分上；对其进行信号通路分析发现差异基因分布广泛,涉及糖、脂类代谢、凋亡、信号通路及各种疾病等等。3.应用Real-time PCR抽检部分差异表达基因,Real-time PCR结果与芯片结果在上调或下调的趋势上一致,且数据较为接近。结论：1.基因芯片是筛选PCOS相关差异基因的一种有效方法,PCOS和非PCOS来源hESCs分化的脂肪细胞在基因表达上有差异,这些差异基因很多涉及糖、脂代谢,并与甾体激素相关,进一步研究后有部分基因可作为PCOS发生发展的候选基因。2. Real-time PCR结果证实本次基因芯片筛查结果真实、可靠。第四部分：NROB2在胚胎干细胞分化的脂肪细胞中的分布与表达目的：分析探讨NROB2基因表达与PCOS之间的关系。以期为PCOS患者早期筛选和有效地治疗提供新的分子标志物,为临床治疗提供新的理论基础。方法：1.实验材料是PCOS来源的hESCs系p-hES-1和非PCOS来源的hESCs系ZZU-hES-1分别定向诱导分化的脂肪细胞。2.在分化不同阶段(0天、7天、14天、21天),行免疫细胞化学检测hESCs分化的脂肪细胞中NROB2基因的产物SHP蛋白的表达和细胞定位。3.应用Western-blotting检测脂肪细胞中SHP蛋白的表达情况。结果：1.SHP蛋白在胞浆中表达,其表达随着脂肪细胞的成熟而增加。2. Western blotting检测,结果提示p-hESC-1组分化的脂肪细胞的SHP量显著高于ZZU-hESC-1组分化的脂肪细胞。结论：1.免疫细胞化学和Western blotting的检测结果与基因芯片的结果相符,验证了SHP蛋白在PCOS来源胚胎干细胞分化的脂肪细胞中也存在高表达。2. NROB2基因可能参与PCOS的发病,并与PCOS患者的肥胖、高胰岛素血症、胰岛素抵抗及糖尿病的发生相关联。更多还原

【Abstract】 Polycystic ovary syndrome (PCOS) is a common disease affects women of childbearing age. Its typical clinical manifestations include infertility, oligo-ovulation or anovulation, hyperandrogenism and so on. The prevalence of overweight and obesity among PCOS patients is 50%. The etiology of PCOS is complex. The pathogenesis is not well explained by the current literatures. And there are no clear and effective treatment and preventive measures. Genetic factor appear to play an important role in the pathogenesis of this disease. Besides, scholars in this research field have paid more and more attention to obesity in recent years. There have been numerous studies showing that adipose tissue play an important role in the development and maintenance of PCOS.Human embryonic stem cells (hESCs) theoretically have the ability to differentiate into every cell types in human’s body. In recent years, a relatively new area in this study is to establish hESCs lines carrying inherent or "transgene" genetic diseases. At present, there has been no successful case of establishing hESCs lines derived from embryos of PCOS patients both at home and abroad. If such cell lines have been established, we might be able to use them to study the pathogenesis of PCOS. And this can avoid many drawbacks of animal experiment. The characteristics of hESCs include unlimited proliferation, pluripotency in vitro and stability of chromosomes. Different batches of human embryonic stem cells can maintain their consistency after differentiation into fat cells. So using hESCs for researches is conducive for manipulation on genetic level and has comparability among groups. It can also avoid the pain caused by taking tissues from patients’ body.At present, there have been many reports of successful differentiation from hESCs to fat cells. But the efficiencies of differentiation were generally low. This becomes a major obstacle to future researches. So it is imperative to improve the differentiation efficiency.In this study, we used PCOS patients’discarded embryos to establish human embryonic stem cell lines, differentiated them into fat cells, and further tested the function of their lipid metabolism. Microarray is an accurate, comprehensive and fast molecular biological method. We used microarray to compare the differentially expressed genes between adipocytes defferentiated from both non-PCOS and PCOS derived hESCs. We screened out the metabolism-related genes from microarray results and studied the selected genes in order to clarify their function in pathogenesis of PCOS.This study consists of four parts as follows:Part I:On the basis of successful establishing hESCs lines of non-PCOS, we built up hESCs lines derived from the PCOS patients’ embryos, and discussed its significance.Part II:We differentiated hESCs into fat cells, and took measures to improve the differentiation efficiency.Part III:After we achieved a relatively high efficiency of differentiation, we applied microarray technology to analyze differential genes between adipocytes differentiated from PCOS and non-PCOS derived hESCs.Part IV:We validated the high expression gene-NROB2 on protein level, and studied its distribution and expression in fat cells which are differentiated from hESCs.Part I:The study of method and significance of establish hESCs lines derived from PCOS patients’embryosObjective: To establish hESCs lines from non-PCOS and PCOS patients’embryos, explore effective method and analyze the impact factor.Methods:1. Fresh or thawed discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university. All patients signed informed consent before experiment. Sequential culture was used for developing these embryos into blastocysts.2. Immunization and mechanical method were used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICMs or the whole embryos on feeder layer. There were three types of them:mouse embryonic fibroblasts feeder layer, human foreskin fibroblast feeder layer or 1:1 mixed feeder layer by the above two types. Purification was done by repeated passage using the mechanical method or collagenase method. After identification of those cells, the hESCs lines were estblished.Results:1. In this study, we collected 924 fresh discarded embryos from 401 non-PCOS patients and 59 fresh discarded embryos from 17 PCOS patients. We also thawed 83 frozen embryos from 25 non-PCOS patients and 19 frozen embryos from 4 PCOS patients. These embryos were sequential cultured to the blastocysts. In total, we got 86 ICMs.2. In this study, we collected discarded embryos and developed them into blastocysts, used immunization and mechanical method to separate ICMs of high-quality blastocysts, planted the ICMs in three different types of feeder layers. The result showed mixed feeder layers can best maintain the stem cells’growth. We also found that the optimal density for inoculation of the mixed feeder layer was 6×105/cm2.2. There were many advantages of using mechanical method passage:clone grew fast with good shape, and wouldn’t easily turn to differentiation. So we used mechanical method for hESCs passage. In total, we successfully established 17 hESCs lines, including 12 non-PCOS and 5 PCOS derived. All of them had been through complete identification.Conclusion: 1. It is essential to choose the well culture system and maintains its stability for the successful establishment of the hESCs lines.2. The rate of establishment of the hESCs lines on mixed feeder layers was significantly higher than on other feeder layers. The efficiency of immunization and mechanical method of isolating ICMs were equivalent. But using mechanical method passage can avoid karyotype variation.3. Establishing hESCs derived from PCOS patients can provided a good in vitro model for study of PCOS early embryo development and the pathogenesis, by screening for candidate genes.Part II:Study on hESCs differentiation into fat cells and differentiation EfficiencyObjective:To investigate the influence that rosiglitazone and bFGF had on hESCs’ differentiation into fat cells, and compare the adipocytes’adipogenic ability between PCOS and non-PCOS groups.Methods:1. The experimental material were hESCs lines which were established by the PCOS patients and non-PCOS patients’embryos in part I, including 3 hESCs lines derived from the non-PCOS patients,3 hESCs lines derived from PCOS patients.2. We added different concentrations of rosiglitazone into the basal differential medium, respectively with 0,1μmol/L, 10μmol/L,100μmol/L of rosiglitazone. We also added different concentrations of bFGF into the basal differential medium, respectively with 0,1 ng/ml,5 ng/ml,25 ng/ml of bFGF. We tried to explore the optimal concentration for rosiglitazone and bFGF.3. Grouping for the induction protocols was done according to the time when rosiglitazone and bFGF were added.4. We tried to figure out the best induction protocol, using adipocyte oil red O staining, triglyceride measurement, MTT assay and RT-PCR to detect the expression of PRARγ-2.5. After finding out the best induction protocol and optimal concentration of rosiglitazone and bFGF, we compared the adipogenic ability of adipocytes of PCOS versus non-PCOS.Results:1. With the extension of differentiation time, scattered mature fat cells could be seen in the second weeks. Then more and more fat cells appeared, with lipid droplets inside. Small lipid droplets in cytoplasm gradually aggregated and accumulated, pushed the nuclei to the other side of the cell. And the shape of adipocytes gradually turned round or oval. The lipid droplets in fat cells could be specifically stained red by oil red O, while undifferentiated cells and non-lipid cells didn’t get colored.2. The triglyceride levels of fat cells can be estimated as the efficiency of fat cell differentiation. 10μmol/L of rosiglitazone induced differentiation had significantly higher efficiency than the other three groups (P<0.05).5ng/ml bFGF induced differentiation had significantly higher efficiency than the other 3 groups (P<0.05). Adding the above-mentioned two kinds of inducing differentiation factor in the latter half of the induction period could achieve more fat cells.3. PCOS derived hESCs adipogenic differentiation capacity was stronger than non-PCOS derived hESCs, but there was no significant statistical difference.Conclusion:1. We set up the experimental model of hESCs and differentiated them into fat cells in vitro of high efficiency. It was shown that 10μmol/L rosiglitazone and 5ng/ml bFGF added at half-cycle could achieve most fat cells.2. The differentiated cells not only have morphology of adipocytes, but also have the function of triglyceride accumulation. These cells provide the material for our studies of fat cell differentiation and metabolism on cellular level.Part III:Differential gene screening on fat cells differentiated from PCOS derived hESCs by microarrayObjective:To explore the molecular mechanisms in pathogenesis of PCOS, using microarray to screen for PCOS-related molecular markers and genes on the prepared adipocytes, so as to provide clues and basis for clinical diagnosis and treatment.Methods: 1. Three hESCs lines derived from PCOS (p-hESC-1, p-hESC-2, p-hESC-3) were directionally differentiated into fat cells as the experimental group 1,2,3. One hESCs line derived from non-PCOS (ZZU-hESC-1)) was differentiated into fat cells as the control group.2. We applied Beijing Boao Crystal Core (?) human whole-genome oligonucleotide microarray to screen for differential genes between fat cells differentiated from PCOS and non-PCOS hESCs lines.3. Real-time PCR was used to validate the differential genes.Results:1. A total of 153 differentially expressed genes between PCOS and non-PCOS derived fat cells were found according to the result of microarray analysis, including 91 up-regulated expression genes and 62 down-regulated expression genes.2. Those genes belonged to different molecular function family, including DNA binding, metal ion binding, hydrolytic enzymes, receptor activation and transfer of enzymes, etc. They participated in several biological processes, including glucose metabolism, lipid metabolism, cell signal transduction, etc, and this part of the differential gene accounted for half of the total. There were still some differential genes in different cell components. Analysis of the differential genes in the signal pathway found that those differential genes are widely distributed, involving glucose, lipid metabolism, apoptosis, the signaling pathway, a variety of diseases and so on.3. Real-time PCR was used to validate some differentially expressed genes. Real-time PCR results were accordant with microarray results in upward or downward, and the data were close to microarray results.Conclusion:1. Microarray is an effective way to screen PCOS-related differential genes. There are several differentially expressed genes between fat cells differentiated from PCOS and non-PCOS derived hESCs. Some differences genes involved in glucose metabolism, lipid metabolism, and related to steroid hormone. Some of them can be served as candidate genes for PCOS occurrence and development after further study.2. Real-time PCR results confirm that this microarray screening results are a true and reliable. PartⅣ:NR0B2’s distribution and expression in the fat cells of hESCs differentiatedObjective:To analyze and discuss the expression of NR0B2 gene and its relationship with PCOS thus to find out a new molecular marker for PCOS and provide a new theoretical basis.Methods:1. The subject was the fat cells differentiated from PCOS derived hESCs lines p-hES-1 and non-PCOS derived hESCs lines ZZU-hES-1.2. In different stages of differentiation (0 days,7 days,14 days,21 days), we detected the expression and localization of SHP (SHP protein is the product of NR0B2 gene) in the fat cells by immunocytochemistry.3. We applied Western-blotting to detect SHP protein expression of fat cells.Results:1. SHP protein expressed in cytoplasm of the adipocytes. Its expression increased with the maturation of the fat cells.2. Western blotting results suggested that protein SHP in p-hESC-1 group was significantly higher than that ZZU-hESC-1 group differentiated into fat cells.Conclusion:1. The results of immunocytochemistry and Western blotting consisted with the results of microarray. It was verified the NR0B2 gene and its product protein SHP was highly expressed in the fat cells defferentiated from PCOS derived hESCs.2. NR0B2 gene might be involved in the pathogenesis of PCOS and associated with obesity of PCOS patients, hyperinsulinemia, insulin resistance and diabetes.更多还原