【作者】 郝小静；

【导师】 杜立新；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 近年来，作为精子和卵子的祖细胞的原始生殖细胞(PGCs)已成为另一种可选择的多能性干细胞。来源于PGCs的干细胞称胚胎生殖细胞。迄今为止仅在小鼠上获得PGCs来源的真正意义上的胚胎干细胞(ES cells)，即具有参与生殖系传递能力的ES细胞。 取孵化5.5～6.5天含PGCs的鸡胚生殖腺及其周围组织，经0.25％胰酶+0.02％EDTA消化后，接种于24孔培养板内。加入新鲜的EG细胞培养液(含DMEM、胎牛血清、鸡血清、β-巯基乙醇、L-谷氨酰胺、HEPES、鸡胚浸出液以及细胞因子等成分)培养24小时以后，PGCs开始部分贴附于共培养的生殖原基细胞上。培养3～4天的PGCs大多已转变为由2～20细胞构成的集落，其中包括从PGCs群集或细胞本身增殖所形成的两种集落类型。培养至5～7天，类EG克隆数目明显增加。类EG细胞克隆呈规则的集落状生长，多层分布，且有明显的花纹。克隆内的细胞间并不紧密排列，因此不难看出克隆内的单个细胞。这与经典的小鼠胚胎生殖细胞的形态有所不同。 原代培养时，我们采用PGCs与相同来源的生殖原基细胞共培养的方式，效果良好。继代培养时，经过反复实验比较鸡胚原代成纤维细胞饲养层(PCEF)、小鼠原代成纤维细胞饲养层(PMEF)、SNL细胞饲养层等三种不同饲养层的作用效果，最终发现以鸡胚原代成纤维细胞制作饲养层同时添加各种生长因子最适合鸡类EG细胞的生长，但PCEF与PMEF之间的差异不显著。本研究中，通过分组添加不同生长因子，结果发现，培养初期不同的分组在培养过程中出现的类EG集落数相差不明显。而随着培养时间的延长，添加LIF、bFGF、SCF、及IGF-1组表现的优势越来越明显，对促进类EG集落的持续增多效果最佳。四种生长因子的添加量分别为：100IU／ml，10ng／m1，10ng／ml，10ng／ml。 在鸡类EG细胞的传代过程中，采用0.25％胰酶与0.02％EDTA的联合作用将集落离散收到了很好的效果。胰酶和EDTA的协同作用，缩短了消化时间，同时降低了消化液对细胞的损伤作用。除酶的作用以外，外源性物质的添加、血清的更换、温度和PH值的改变等因素对鸡类EG细胞的分离培养均有重要的影响。 研究中分离得到的鸡类EG细胞经PAS染色后呈深紫红色，而鸡胚成纤维细胞并不着色。将消化分散的类EG细胞在无分化抑制因子的类EG培养基中重悬培养，3～4天后，可见部分细胞团形成简单的胚体。在饲养层上生长的类EG细胞集落，在去除抑制分化因子的条件下培养，长时间不传代，可见细胞生长缓慢，自发分 山东农业大学硕士研究生论文（2003）化，首先表现为失去原有的形态，进而与周围细胞界限不清，集落外围的细胞与饲养层连成一片。最后将传至3代、4代的类EG细胞集落离散后制作嵌合体，得到了一只毛色嵌合体鸡。 本研究分离得到的类EG细胞经形态学鉴定、PAS染色、体外分化实验和嵌合体鸡的制作等实验证明其具有胚胎干细胞的诸多类似特性。类EG细胞所具有的多能性、定向整合及较高的嵌合能力，对高效培育鸡的新品种有重大的意义。更多还原

【Abstract】 Recently, primordial germ cells(PGCs), which are the progenitors of the sperm or EGg cells, have provided an alternative source of pluripotent stem cells. The stem cells derived from PGCs are called embryonic germ cells(EG cells). To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem cells(ES cells).Gonadal primordial germ cells were got by isolating embryonic gonads and surrounding tissues from chicken embryos(5.5-6.5 days of incubation) and dissociating the tissues by standard trypsinization procedure(0.25%Trypin+0.02%EDTA). The EG cell culture media consisting of DMEM medium supplemented with FBS, chicken serum, beta-mercaptoethanol, L-glutamine. HEPES, chicken embryonic extract and cytokines etc. After 24 hours culture, the isolated PGCs were selectively attached on the gonadal stromall cells in the plates. However, at 3~4 days primary culture, almost all the PGCs were formed of 2~20 cell mass. After 5~7 days, the numbers of putative EG clonies increased distinctly. These clonies were uniformly round, multi-layered and well delineated. The cells did not pack strongly together in small clumps and it was not difficult to discern the individual component cells. This morphology was slightly different from that of mouse EG cells.At primary culture, PGCs co-cultured with their gonadal stromall cells were well grown . When subculture, we used primary chicken embryonic fibroblast (PCEF), primary mice embryonic fibroblast (PMEF) and SNL cells to make feeder cells. Forward research founded that the PCEF cells were the most suitable for the growth of putative EG cells when having various cytokines. In this study, the putative EG cell culture media was supplemented with LIF, bFGF, SCF and IGF-1. As a result, the idealdose of LIF, bFGF, SCF and IGF-1 was: 100IU/ml,10ng/ml, 10ng/ml, 10ng/ml. In addition, there were still many other factors were influencing on the culture of putative EG cells such as the density of FBS, the cell-dispersed liquid, the temperature and PH of the culture environments etc.In this study, the chicken putative EG cells continued to- be PAS positive and manifested some typical morphology. Under suspension culture without cytokines, the putative EG clonies could be induced to form simple embryoid bodies. After long-time culture, these bodies began to differentiated into various cell types. Moreover, chimeras when injecting of the cultivated cells into receptor’s x stage blastdermal also showed that the cells were some pluripotent. These study may be useful to the production of transgenic chickens by genomic modification.更多还原