【作者】 欧阳琦；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 干细胞工程学， 2010， 博士

【摘要】 人胚胎干细胞(Human Embryonic Stem Cells, hESCs)是从囊胚内细胞团(Inner Cell Mass, ICM)分离得到,能够在体外无限增殖并且保持核型稳定,在特定的诱导条件下还可以向三个胚层的任何细胞类型分化。因此,hESCs不仅是研究人类胚胎发育的最佳模型,也是未来再生医学应用上极富前景的种子细胞来源。但是hESCs及其分化后的功能细胞表达主要组织相容性抗原(Major Histocompatibility Complex, MHC),移植到宿主体内后可能因MHC不相容而刺激机体引起免疫排斥反应,导致移植治疗失败,这将成为hESCs未来临床应用的主要挑战之一。因此,要实现hESCs及其功能产物安全应用的关键之一就是如何解决免疫排斥问题。本研究主要从两个方面探讨克服干细胞移植的免疫排斥障碍的策略：建立胚胎干细胞库进行MHC配型；建立孤雌胚胎干细胞系,一方面更容易得到HLA纯合的胚胎干细胞系从而提高干细胞库的MHC配型效率,另一方面也是女性患者自身特异性的多能性干细胞。全文共包括2个部分,分述如下：第一章中国人类胚胎干细胞库的建立以及HLA配型分析目的：探讨能否利用质差或者异常受精的胚胎建立胚胎干细胞库,以及建立的胚胎干细胞库用于干细胞移植时HLA匹配的可行性。方法：利用体外受精-胚胎移植(IVF-ET)治疗中的废弃胚胎建立人类胚胎干细胞库,包括：异常原核的胚胎、受精后第三天观察质量较差的胚胎、植入前遗传学诊断异常(PGD)的胚胎。对建立的胚胎干细胞系进行细致全面的特性鉴定以及HLA和ABO基因分型检测。然后与中华骨髓库湖南分库中随机选择的5236名正常人群进行HLA匹配分析。结果：共采用692枚囊胚,建立了188个hESCs系,这些细胞系均具有胚胎干细胞的典型特征,其中153(81.4%)株具有正常二倍体核型。具有二倍体HLA基因型的174株hESCs系与5236名假想患者进行了HLA配型分析,结果表明：这些hESCs系能够为24.94%-56.26%的假想患者提供良好的HLA匹配,并且匹配率的高低与民族差异无关。此外,8个HLA纯合型hESCs系贡献了细胞库不同HLA匹配水平上50-80%的匹配效率。结论：利用临床废弃胚胎能够建立人胚胎干细胞库,并且能够具有较大的HLA配型价值,为解决干细胞移植的免疫排斥问题提供了一个良好的策略。第二章孤雌胚胎干细胞的建立以及与正常胚胎干细胞生物学特性的比较研究目的：通过孤雌激活的方法建立患者特异性胚胎干细胞系,并与正常受精来源的胚胎干细胞进行生物学特性的比较研究,探讨孤雌胚胎干细胞是否与正常受精胚胎干细胞一样具有巨大的临床应用前景。方法：利用人工孤雌激活的胚胎以及IVF-ET过程中自然孤雌激活的1PN胚胎建立孤雌胚胎干细胞(Parthenogenetic embryonic stem cells, pESCs)系,对pESCs系进行细致全面的干细胞特性鉴定。并从三个方面对pESCs系与正常受精hESCs系进行比较研究,包括：印记基因的表达、全基因组范围内基因表达差异的分析、全基因组SNP的分布特点以及三个胚层主要器官发育关键基因的表达对比分析。结果：pESCs仅表达母源性印记基因,父源性印记基因沉默,而正常受精hESCs同时表达父源和母源印记基因。在全基因组SNP分布方面,自然激活孤雌胚胎来源的chHES-32表现为绝大多数位点纯合,人工激活孤雌胚胎来源的chHES-69表现为大片段的纯合与杂合并存,而正常受精胚胎的chHES-8和chHES-10表现为绝大多数位点杂合。将pESCs和正常hESCs制备EB,体外自发分化21天后,通过RT-PCR以及Real-time PCR检测三胚层主要器官发育关键基因的表达,发现pESCs没有明显的分化缺陷。基因芯片聚类分析发现,pESCs与正常hESCs之间没有明显差异。结论：pESCs在培养过程中能够保持母源性印记基因的表达特点,不同孤雌激活方法得到的pESCs与正常受精hESCs在全基因组SNP的分布上存在明显差别,pESCs没有明显的分化能力障碍。更多还原

【Abstract】 Human embryonic stem cells (hESCs), which are derived from the inner c ell mass of the blastocyst, can proliferate indefinitely in culture, have a normal karyotype, and are able to differentiate into all cell types under the appropriate conditions. Therefore, they represent both a valuable tool for the study of human development and a highly promising source of cells for future regenerative medicine technologies. However, a major challenge for the development of these therapeutic technologies is that transplantation of hES-cell derivatives can lead to allograft rejection due to major histocompatibility complex (MHC) incompatibility. A key issue for safe therapy from hESCs and their derivatives is how to overcome the immune barrier to stem cell transportation. In our study, we estimate two strategies for avoiding immunological rejection. One is to establish a hESCs bank with a relatively large number of hES cell lines for HLA-matching, the other is to produce parthenogenetic embryonic stem cells that is benefit for improving the HLA-matching rate of our stem cell bank and is also personalized pluripotent stem cell line for female patients. The thesis consists of two parts as following: Chapter 1:HLA-matching potential of an established human embryonic stem cell bank in ChinaIntroduction:It was suggested by theoretical calculation that establishment of 150-170 human embryonic stem (hES) cell lines with various human leukocyte antigen (HLA) genotypes will help to prevent allograft rejection in hES cell based transplantation therapy. However, whether embryos from In Vitro Fertilization treatment could provide sufficient HLA diversity for matching is unclear and ethical debate will arise considering destroying large number of normal human embryos for derivation.Methods:Blastocysts with various abnormalities, including abnormal pronuclear zygotes, poor quality day 3 embryos after fertilization and abnormal embryos after pre-implantation genetic diagnosis were used to establish a hES cell bank. Detailed characterizations were conducted on most of the established hES cell lines. Then the HLA and ABO blood group genotypes were analyzed and further matched to that of 5236 randomly selected local individuals from China bone marrow bank.Results:From 692 blastocysts used,188 hES cell lines were established and 156 (83%) have euploidy karyotype. These cell lines showed typical hES characters in performed analysis.174 cell lines with diploid HLA genotype were used for HLA matching and provide a beneficial match for 24.94%-56.26% of the 5236 presumed patients, regardless of ethnic difference. Eight hES cell lines with homozygous HLA genotypes from the bank contribute most to the matching rate in a range of 50-80% in different mismatch level.Conclusion:A hES cell bank with great HLA-matching value could be established using only the clinical invaluable embryos.Chapter 2:Establishment of human parthenogenetic embryonic stem cells and comparison with normal embryonic stem cellsIntroduction:To establish personalized pluripotent stem cell lines through parthenogenesis, and then to estimate whether the clinical application potential of pESCs is equal to that of normal hESCs through comparing the biological characterization with normal hESCs.Methods:pESCs were derived from embryos by parthenogenetic activation of human oocytes or naturally parthenogenetic activation of 1PN embryos in IVF-ET process, and were fully characterized in stem cell property. Furthermore, pESCs were compared with normal hESCs in four aspects, including:the expression pattern of imprinted genes, the analysis of differential genes expression in genome, genome-wide SNP genotyping, and the key genes expression related with the development of main organs from all three germ layers.Results:pESCs only expressed maternal imprinted genes and the paternal imprinted gene expression was absent, while both paternal and maternal imprinted genes were expressed in normal hESCs. For genome-wide SNP genotyping, chHES-32, which is derived from 1PN embryos, is a homozygous diploid parthenote due to duplication of a single haploid pronucleus; unlike the chHES-69, which was created by prevention of second-polar-body extrusion, resulting in partial heterozygosity due to the failure to segregate the rearranged chromosomes at meiosis II. In contrast, the normal hESC lines-chHES-8 and chHES-10 are highly heterozygous and do not exhibit pericentromeric homozygosity pattern on the chromosomes, as observed in pESCs. pESCs and normal hESCs were detached to grow as aggregates in suspension for 21 days to start up differentiate, and then both two kinds of EBs were tested the expression of the key genes expression related with the development of main organs from all three germ layers by RT-PCR and Real-time PCR. The results showed that no common differentiation defects are found in pESCs. We did clustering analysis of gene-chip results and found that there are no distinct differentiation between pESCs and normal hESCs.Conclusion:pESCs can maintain the expression pattern of imprinted genes in culture, the genome-wide SNP genotyping is obviously different between the normal hESCs and pESCs from different parthenogenetic activation methods; no distinct differentiation defects are found in pESCs.更多还原