【作者】 赵会敏；

【导师】 卢克焕；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 精原干细胞(spermatogonial stem cells, SSCs)位于动物睾丸的精细小管,是动物体内唯一一种定向分化为精子的成体干细胞。分离、纯化和体外培养SSC,可以为SSC的体外研究提供必要的实验材料。定向诱导SSC分化成精子可以研究精子发生的机理,同时也可以为无精症患者提供生育自己后代的机会。通过转染SSC,诱导其分化成携带有外源基因的精子细胞或精子,利用这些携带外源基因的精子细胞或精子受精就可以产生转基因动物。这种转基因胚胎产生的过程和自然条件下精子、卵子结合产生胚胎的过程一样,不涉及体细胞重编程过程,所以利用SSC生产转基因动物的效率要远远高于体细胞核移植法。本论文的研究内容是巴马小型猪SSC的分离、纯化、鉴定、体外长期培养以及转基因的研究,其结果如下：1.巴马小型猪睾丸的形态学观察和精原干细胞的鉴定本研究首先对巴马小型猪睾丸组织进行了形态学观察和SSC的鉴定。组织学观察结果发现1月龄仔猪睾丸的SSC还没有分化,而2月龄仔猪睾丸中已经有精子产生,所以1月龄仔猪睾丸适合于SSC的分离。已有的研究证明猪的精原干细胞表达UCHL1,并且可以和植物凝集素DBA结合,SSC的免疫组织化学鉴定结果发现巴马小型猪SSC表达UCHL1和CDH1,可以和植物凝集素DBA结合,但不表达C-KIT,从而证明抗体UCHL1、CDH1以及植物凝集素DBA是巴马小型猪SSC的生物标志物,可以用来鉴定体外培养的SSC。所以,本研究选用抗体UCHL1、CDH1和植物凝集素DBA用来鉴定体外培养的精原干细胞。2.精原干细胞的分离和体外培养本实验采用组合酶消化法将巴马小型猪睾丸组织消化成分散的细胞,培养60 h,扩增SSC的数量,然后通过差异贴壁法分选出SSC,将分离出的细胞接种到STO饲养层上进行培养。在培养的前两代,由于细胞的数量少,不能形成细胞簇,从第三代开始,很多细胞聚集在一起形成细胞簇,这些细胞簇的细胞结合不够紧密,边缘不清晰。目前为止,SSC在体外已经传代达30代,维持了细胞簇形成的特性。3.体外培养的精原干细胞的鉴定免疫荧光染色结果显示这些细胞簇表达SSC生物标志物UCHL1和CDH1,并且可以和植物凝集素DBA结合,细胞簇中部分细胞表达OCT4; RT-PCR检测结果证明这些细胞簇表达基因nanog、sox2、vasa、uchl1和thy1,鉴定结果证明这些细胞簇是由SSC组成。在体外培养的SSC中存在着大小不同的UCHL1阳性细胞,说明SSC大小本身存在着差异。SSC超微结构观察发现体外培养的SSC的线粒体脊比组织内SSC线粒体脊丰富。DNA含量分析显示部分巴马小型猪SSC在体外长期培养后经流式细胞仪分析发现单倍体峰。4.精原干细胞转基因方法的初步探索本研究尝试将外源基因导入到SSC中。首先尝试利用脂质体转染法将携带有绿色荧光蛋白基因的质粒转染到SSC中,但结果发现这种转染途径效率低,不能得到阳性细胞簇。之后,我们尝试利用慢病毒感染法将外源基因导入到SSC内,实验发现在MOI(multiplicity of infection)值为50时,可以有效的将GFP基因导入到SSC内,可以得到阳性细胞簇。综上所述,本研究分离纯化了巴马小型猪SSC,这些SSC可在体外培养到30代以上,为精原干细胞的基因操作和精子分化机理的研究提供了充足的实验材料。本研究还发现巴马小型猪SSC在体外长期培养后经流式细胞仪分析发现了单倍体峰,利用慢病毒感染法可以有效的将GFP基因导入到SSC内。这些研究结果为转基因巴马小型猪的生产建立了实验平台,也为其他动物SSC的分离、培养、体外诱导以及转基因动物的生产提供理论和技术支持。更多还原

【Abstract】 Spermatogonial stem cells (SSCs), which locate on the basement membrane of the seminiferous tubule in testis, are the unique stem cells which can differentiate and produce spermatids. Isolation, purification and proliferation of the SSCs in vitro will provide necessary material to study SSCs in vitro. Induction of SSCs and production of spermatid in vitro can show us the mechanism underlying the spermatogenesis. Fertilization with the spermatids induced from transgenic SSC will be an efficient approach to produce transgenic animals.This study focused on isolation, purification, identification, long-term proliferation and transfection of Bama mini-pig SSC and the results showed below:1. Morphology observation of testis tissue and identification of SSC in the tissue.Morphology observation of testis tissue showed that no spermatocyte, spermatid was observed in the slices of the testis at the age of 1 month, while they were observed in the slices at the age of 2 months, this result shows that the testis at 1 month of age is suitable to isolate SSC. Immunohistochemical staining showed that the SSCs in testis at 1 month of age expressed UCHL1, CDH1 and could bind with lectin DBA (Dolichos biflorus agglutinin).Thus lectin DBA and antibodies including UCHL1 and CDH1 were employed to identify SSCs of Bama mini-pig cultured in vitro.2. Isolation and long-term culture of SSC.The testis tissue was digested and dissociated with enzyme, SSCs were purified using differential plating and cultured on STO feeder layer in serum-free medium. At the first two passages, no cluster was observed, at the third passage, the cells aggregated together and formed clusters with unclear border. The SSCs have been cultured for 30 passages to date and kept cluster formation ability.3. Identification of the SSCs cultured in vitro.Immunocytochemical staining showed that the clusters could bind with DBA and expressed UCHL1 and CDH1, some cells in the colonies expressed OCT4, The result of PT-PCR showed that the clusters expressed nanog, sox2、vasa、uchll and thyl, which showed that the clusters were formed with SSCs and named spermatogonial stem cells cluster. Meanwhile, different sizes of UCHL1 positive cells were observed in the cultured cells, which showed that the size of SSCs was various. Ultrastructure observation showed that cristaes of mitochondria in SSCs cultured in vitro were more abundant than those in tissue. Haploid peak was found in DNA content analysis of the cultured cells in different passages.4. Transfection of SSCs cultured in vitroIn this study, two methods were tried to transfer exogenous gene into SSCs. Firstly, lipofectamine LTX was used to transfer the plasmid into SSCs, while the result showed that this method was not effective. Lentivirus, which expressed EGFP, was also used to infect SSCs, the result showed that the SSCs could be transduced effectively when MOI was set at 50 and transgenic SSCs were observed.In conclusion, SSCs of Bama mini-pig were isolated and long-term cultured in vitro, which provided sufficient cells for transgenic research or exploring the mechanism of the spermatogenesis. In this study, Haploid peak was found in DNA content analysis of the cultured cells in different passages, transgenic SSCs were yielded by lentivirus infection, which established a platform to produce transgenic Bama mini-pig and provided experience for SSCs study of other domestic animal.更多还原