【作者】 周冠月；

【导师】 李碧春；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 精原干细胞(spermatogonial stem cells, SSCs)是雄性生殖细胞的前体细胞,具有终生扩增性和永生性,是产生雄性生殖细胞的保证,也是生殖系中唯一的在成体中还能增殖的细胞。目前,SSCs已成为发育学研究的热点之一,并在兽医临床学和转基因动物的制备方面具有广阔的应用前景。本研究分离提取了孵化至第19d的鸡胚睾丸中SSCs进行冷冻保存和体外培养,以期筛选出适合鸡胚SSCs冷冻保存的适宜的冷冻保护剂及其浓度;同时进行了体内鸡SSCs转染外源基因(pEGFP-N1)的探索,观察了转染后外源基因在精子、种蛋胚盘及不同孵化日龄胚胎及其组织器官中的表达,为以后利用SSCs生产转基因鸡的研究奠定基础。研究结果可归纳为以下几个方面:1.本研究采用二酶三步法获取孵化第19d的鸡胚SSCs,分别单独采用浓度为5%、10%、15%的二甲基亚砜(DMSO)、乙二醇(EG)、甘油作为冷冻保护剂进行超低温冷冻保存,复苏后台盼兰染色检查细胞存活率,比较不同浓度的各种冷冻保护剂对鸡胚SSCs的冷冻保存效果;并在添加细胞因子的体外培养体系中,观察复苏后SSCs存活和增殖。结果显示:(1)当DMSO的浓度为5%、10%及15%时,复苏后细胞存活率分别为73.1%、88.6%和74.8%,三者差异显著(P<0.05);当乙二醇浓度为5%、10%和15%时,复苏后细胞存活率分别为69.4%、83.1%和65.2%,三者差异显著(P<0.05);当甘油浓度为5%、10%、15%时,复苏后细胞存活率均小于15%,三者差异不显著(P>0.05)。(2)以10%DMSO为冷冻保护剂进行冷冻保存的SSCs,复苏后接种在以鸡胚胎成纤维细胞为饲养层的培养体系中进行体外培养,发现复苏后生长良好并聚集成集落,AKP染色呈阳性反应,在无饲养层的培养体系中没有集落生成;而以5%和15%的DMSO为冷冻保护剂的SSCs,无论饲养层存在与否均无集落生成;以5%、10%、15%乙二醇为冷冻保护剂时,复苏后无论饲养层存在与否,SSCs均能增殖,但未有AKP阳性集落生成;以5%、10%、15%更多还原

【Abstract】 Spermatogonial stem cells (SSCs), as the originated cells of germs in male, which was immortalized and could be proliferate in the whole developmental process. Up to date, SSCs is a focus in the field of developmental biology, which has widely prospects of application in clinical of veterinarian and producing transgenic animals. In this study, the SSCs from chickens’embryos hatched for 19 days were isolated for cryopreservation, so as to search for suitable cryopresevation system for SSCs . At the same time, SSCs were transfected by exogenous DNA(pEGFP-N1) in vivo, and the expression of pEGFP-N1 were detected in sperm, embryo discs and hatching embryos in different periods, tissues, which provide the basement for producing transgenic chickens. The results were showed as following:1 The effect of Dimethylsulphoxide (DMSO) , ethylene glycol (EG) and glycerin(Gly) cryoprotectants, each at (5%, 10%, and 15%) concentration on the 19d chicken embryonic SSCs was investigated. The results indicated that (1) When the concentration of DMSO were 5%, 10% and 15%, the viability rate were 73.1%, 88.6% and 74.8% respective, and the difference were significant(P<0.05). When the concentration of EG were 5%, 10% and 15%, the viability rate were 69.4%, 83.1% and 65.2% respective, and the difference were significant(P<0.05). When the concentration of glycerin were 5%, 10% and 15%, the viability rate were less than 15%, and difference were not significant (P>0.05). (2) The SSCs, 10% DMSO as freezing media, were cultured on the chicken embryos cells as layer cells after thaw, then found the SSCs surviving well and forming colony which were positive after AKP staining, but when the SSCs were cultured without layer cells, they couldn’t form colony. When the SSCs, 5%, 10% and 15% EG as freezing media, were cultured after thaw, then they could proliferate, but couldn’t form colony with or without layer cells. When the SSCs, 5%, 10% and 15% glycerin as freezing media, were cultured after thaw, then they survived about 12h. The results showed that 10%DMSO were the best freezing media for the 19d chicken embryo更多还原