【作者】 孙燕；

【导师】 李文鑫；

【作者基本信息】 武汉大学 ， 微生物学， 2003， 博士

【摘要】 早期胚胎是细胞生长、发育、分化最旺盛时期，大多数基因都处于活化状态，是发现和分离有重要功能新基因的极好材料。尽管小鼠等模式动物，对于我们认识哺乳类动物早期胚胎发育提供了许多重要信息，但它与人类存在着必然的差异。为此，我们根据实际情况和条件选择人早期胚胎为材料，进行人早期胚胎中表达的新基因的分离和功能研究。 在构建早期人胚胎cDNA文库的基础上，应用杂交筛选和表达标签（EST）分析技术从3-5周龄人胚胎cDNA文库中得到一个克隆子L30，应用5’RACE技术，克隆了其全长cDNA。经过对全长cDNA序列的测定及其结构特征的分析，证实L30为一个新的锌指蛋白基因，国际命名委员会命名其为ZNF268。为了研究ZNF268的生物学功能，我们分别在mRNA和蛋白质水平研究了ZNF268在不同器官和组织中的表达图谱，为进一步深入研究奠定基础。 以³²P标记的ZNF268cDNA特异片段（3’UTR）作为探针，通过Northern杂交技术，检测了ZNF268在42种正常人成体器官和组织以及8种晚期胎儿器官中的表达。结果显示，除阳性对照外，检测的所有样品中均无明显的杂交信号。同时，从早期人胚胎（3-5周龄及3月龄）提取总RNA与ZNF268探针进行杂交，均得到了3.8kb的杂交条带。Northern结果表明ZNF268mRNA仅特异性表达于早期人胚，而在成人组织中不表达。 为了进一步在翻译水平研究ZNF268蛋白的表达，我们构建了ZNF268重组原核表达载体，纯化在大肠杆菌中表达的产物，制备了特异的zNF268抗体paZNF268ab。以paZNF268ab抗体为依托，用Western印迹法检测出在4月龄人胚胎肝脏组织中，有分子量为108KDa的ZNF268蛋白的特异表达信号。进一步利用免疫组织化学方法检测ZNF268蛋白在5周至7月龄人胚肝中的表达。结果显示ZNF268蛋白在不同发育时期的人胎肝中表现出不同的表达水平，其表达量随着胚龄增加、即胚肝分化发育成熟而下降。由此可见ZNF268可能参与调控早期人胚肝的分化和发育。免疫组织化学的结果还显示，ZNF268在早期的造血干、祖细胞中表达。因此ZNF268也可能参与早期造血细胞的分化发育，从而与早期人胚造血相关。 大多数锌指蛋白做为转录调控因子，在细胞中的表达集中于细胞核中。而用免疫组织化学方法检测ZNF268蛋白表达产物的定位时，发现ZNF268蛋白集中于细胞质中。为了进一步确认ZNF268蛋白在细胞中的表达定位，构建了与绿色荧光蛋白融合的部分及全长ZNF268表达载体，对在COS7细胞中表达的不同ZNF268片段融合EGFP的蛋白质进行细胞内定位。结果证实ZNF268蛋白的表达主要定位于细胞质中。这一结果表明ZNF268不同与大多数的锌指蛋白，其发挥功能可能是通过胞质内的调控途径。同时这一结果为确定ZNF268的靶向物及研究ZNF268的功能奠定了基础。 在研究ZNF268表达产物的细胞内定位时，我们还观察到ZNF268KRAB盒的表达产物定位于细胞核中。CZHZ型锌指蛋白的KRAB盒具有和其它因子相互作用而介导转录抑制的功能。ZNF268的KRAB盒保守性很高，可能同样是一个转录抑制因子。细胞内定位的结果显示ZNF268的KRAB盒主要集中于细胞核中，这与其它转录抑制因子定位于细胞核中是一致的。为了研究ZNF268KRAB盒的功能，构建了一系列含有ZNF268KRAB盒不同片段的真核表达载体;与含有CAT基因的报告载体共转染细胞，分析ZNF268KRAB盒的转录抑制活性。结果证实ZNF268的KRAB盒确实具有转录抑制作用;并且KRAB一A是一个强抑制子，KRAB一B对KRAB一A有协同作用。 在确证了ZNF268有转录抑制功能的基础上，我们希望寻找ZNF268功能作用的靶向物。采用酵母双杂交技术，用ZNF268蛋白作为“诱饵”，筛选18²4周龄（约4一6月龄）的人胚肝文库。经过营养缺陷型筛选、X一a一gal和X一p一gal筛选、及体外免疫共沉淀验证，得到一个与ZNF268蛋白相互作用的蛋白质AHSG。研究表明，AHSG参与人胚肝的发育。ZNF268蛋白与AHSG蛋白的互作说明，ZNF268可能通过与AHSG协同作用而参与调控人胚肝的发育。同时，AHSG不仅仅表达于肝脏，在骨髓生血基质中也有表达，并与多种血液疾病相关，提示ZNF268潜在的与早期人胚造血的关系，以及在某些特定情况下可能与某种疾病相关。 总之，本研究从转录和翻译水平分析了ZNF268在人早期胚胎中的表达特征;研究了ZNF268的细胞内定位并分析了其作用的靶向物。研究结果初步阐明了这一新的锌指蛋白基因在人早期胚胎中的功能。更多还原

【Abstract】 Embryogenesis is a complex process that requires accurate temporal and spatial regulation of gene expression and function. Alteration of this regulation may lead to congenital abnormality and inheritable genetic diseases. Although various animal models have provided some information of early mammalian development, vital differences between human and other animals argue for studies on human development.In an effort to identify and functional characterize genes important for human development, we previously constructed a cDNA library from 3-week-old human embryos. By using a differential hybridization strategy, we were able to isolate, from this library, many genes expressed in the early human embryos, among which, a novel zinc finger gene, ZNF268.To determine whether ZNF268 is involved in human embryogenesis, we first analyzed its expression by Northern blot hybridization on RNA from human embryos and adult human tissues. The ZNF268 probe detected an mRNA of 3.8 kb in length in human embryos. While the mRNA was present in all embryos analyzed, but no ZNF268 mRNA was detectible in all adult human tissuesanalyzed. Those imply ZNF268 play a role specifically during early embryogenesis.To investigate the expression of ZNF268 protein, we first generated an anti~ZNF268 antibody specifically. Western blot analysis indicated that ZNF268 was expressed in the fetal liver, but not in the heart, kidney, brain and spleen. To further investigate ZNF268 expression in development, immunohistochemical analysis was carried on tissue sections of human embryos of various ages. Again, the anti-ZNF268 antibody specifically detected the protein in the liver of a human embryo as early as 5 week old but not in other areas of the embryo. The protein level in the liver hepatocytes remained high up to 4 month old but drastically reduced by 6 month old and to non-detectible levels by 7 months. These results suggest that ZNF268 is involved in the development of fetal liver in early human fetuses. In addition, Immunohistochemistry analysis also indicated that ZNF268 expression was found in the hematopoietic stem/progenitor cells. This make a suggestion that ZNF268 may involve in the development of the blood cells in early human embryo and may have a function in early human hematopoiesis.The immunohistochemical analysis of ZNF268 expression in embryonic liver also revealed, at high resolution, that the protein was localized in thecytoplasm of hepatocytes through out development (5 weeks to 4 months). To further investigate the subcelluar distribution of ZNF268 protein, we expressed the fused EGFP zinc finger protein in COS7 cells. Examination result showed that ZNF268 was predominantly, if not exclusively, a cytoplamic protein, belonging to the small group of cytoplamic zinc figure proteins but distinct from other krtlppel-Iike zinc finger proteins.To examine whether the ZNF268 KRAB possesses transcriptional represser activity, KRAB-A, KRAB-B and KRAB-AB were fused in-frame to the GAL4 expression vector pBXGl respectively. Then they were co-expressed with reporter plasmid pG5SV-BCAT which containing five GAL4-binding elements. Both KRAB-AB and KRAB-A decreased the expression level of the reporter gene in a dose-dependent manner. The KRAB-B suppressed transcription of the reporter gene not as significant as the other two; KRAB-AB repressed the reporter gene distinctly than KRAB-A. In conclusion, KRAB domain of ZNF268 has transcription repressing activity, and ZNF268 may act as a represser.To study the interact protein of ZNF268, we constructed the "Bait" with ful 1 cDNA of ZNF268, and screened in the human liver cDNA library of 18-24 weeks. Now we identified a interact protein AHSG (alpha2-Heremans Schmid Glycoprotein). AHSG mayplay roles in the development of liver, brain and may involve in bone mineralization and in the immune response. The interaction between ZNF268 and AHSG indicate that AHSG, recruiting ZNF268, play a role in early liver development. Otherwise, AHSG also expressed serum, bone marrow hemopoieti更多还原