【摘要】 家禽的原始生殖细胞(primordial germ cells,PGCs)是形成精子和卵子的前体细胞,可在性成熟的家禽体内生成精子和卵子,因此家禽PGCs可应用于以PGCs为基础的现代保种技术、转基因技术以及家禽的发育生物学等研究领域,具有重要的科研与应用价值。本研究使用Nycodenz密度梯度离心法在孵化至13~15期的鸡(Gallus gallus)胚血液中分离纯化鸡胚的PGCs,经显微注射针分拣后,PGCs的纯度可达99%以上,细胞直径主要集中于13~15μm;将所分离纯化的PGCs分别通过碱性磷酸酶(alkaline phosphatase,AKP)染色、高碘酸希夫氏(periodic acid-schiff,PAS)染色及阶段特异性胚胎表面抗原-1(stagespecific embryonic antigens-1,SSEA-1)免疫化学染色进行鉴定,染色结果均出现PGCs所特有的反应;使用活细胞荧光染料PKH26对分离纯化的PGCs进行标记,成功标记的PGCs在荧光显微镜下可观测到红色荧光,将约200个标记后的PGCs,通过腹主动脉注入孵化至14~16期的受体鸡胚中,在孵化至第8天的鸡胚性腺冷冻切片中,可检测到供体PGCs的存在,这一结果表明,所分离纯化的PGCs可定植于受体鸡胚的性腺中。本研究为以PGCs为基础的种质资源保存研究以及转基因技术研究提供了基础资料。更多还原

【Abstract】 Avian primordial germ cells(PGCs), as precursor of sperm and eggs, can develop to sperms and eggs in the sexually matured avian. Based on this property, PGCs can be used in the fields of PGCs preservation, transgenic avian preparation and developmental biology and will play very important role in the fields of scientific research and practical application. In this study, the PGCs from blood of chicken(Gallus gallus)embryos which had been hatched to Hamburger-Hamilton stage 13~15(HH13~15) were separated and purified by Nycodenz density gradient centrifugation. The purity of the PGCs was to 99% and the diameter of the purified PGCs was about 13~15 μm after manually selected by microinjection glass needle. The purified PGCs were identified by alkaline phosphatase(AKP) staining, periodic acid- schiff(PAS) staining and stagespecific embryonic antigens-1(SSEA-1) immunohistochemical staining to show the specific reaction of PGCs.The purified PGCs were labeled with fluorescent dye(PKH 26) and successfully labeled PGCs showed red fluorescence under fluorescent microscope. About 200 labeled PGCs were microinjected into the dorsal aorta of recipient chicken embryos which had hatched to HH14~16. After 8 days of incubation, the gonads of recipient chicken embryos were then separated under a stereoscope and embedded in Neg-50 frozen section medium and frozen in liquid nitrogen. Frozen sections were prepared and observed under fluorescence microscope, the labeled donor PGCs could be observed in the gonad of the recipient chicken embryos, which indicated that the purified PGCs were capable of migrating and settling in gonads of the recipient embryos.This study will lay a foundation for the study of PGCs based germplasm preservation and transgenic technologies.更多还原