【作者】 杨继建；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 硕士

【摘要】 本研究以山羊为材料,对影响山羊ES 细胞、EG 细胞分离与克隆的影响因素进行了探讨。为进一步建立山羊ES、EG 细胞系奠定了基础。实验主要结果如下: 1. 收集妊娠20~55d 山羊胎儿50 例,使用DMEM(高糖)+ 15% NBS(或FBS) +10 ng/mL+ 0.1 mmol/mL?-巯基乙醇+ 2 mmol/mL 谷氨酰胺+ 0.01 mmol/mL 非必需氨基酸+ 100 IU/mL青霉素+ 100 IU/ml链霉素培养液从生殖嵴(腺)中分离克隆山羊EG细胞,43 例出现EG 样细胞集落,最高一例传至6 代。胎龄为30～36 d山羊胎儿最适合山羊EG 细胞分离与克隆,胎龄大于42d,很难得到山羊EG 细胞。从新生关中奶山羊睾丸中分离培养出山羊睾丸支持细胞。对比分离得到的PGCs在饲养层GSCs、MEF、GEF 上的生长效果,发现GSCs>MEF>GEF。在基础培养液中添加10%(1000IU/ml)LIF更有利于山羊EG 的克隆。2. 用全胚法从192 枚关山羊胚胎中分离得到82 枚增殖ICM,接种在MEF 或GEF饲养层上,使用DMEM(高糖)+ 15% FBS + 0.1 mM?-巯基乙醇+ 0.01 mM 非必需氨基酸+ 10 ng/ml LIF + 100 IU/mL 青霉素+ 100 IU/ml 链霉素培养液,用中浓度消化液(0.125% Trypsin+0.02% EDTA)消化效果好,有一枚胚胎传至第8 代。3. MEF、GEF 在山羊胚胎贴壁率无显著差异(P>0.05);MEF 和GEF 饲养层均能促进山羊ES 细胞体外增殖。随着胚龄的增加,胚胎的贴壁率和ICM 的增殖率增加。孵化囊胚的贴壁率和增殖率最高,达90%和60%,与桑椹胚相比,差异均显著(P<0.05);添加LIF(10 ng/ml)有利于山羊ES 细胞(P<0.05)的分离与克隆。4. 山羊ES 细胞对胰酶不是很敏感, 0.125% Trypsin+0.02% EDTA 是较好的ES 细胞消化液,对细胞综合损伤力小,且传代后ES 细胞集落形成能力也较高。6. 分离得到的山羊ES 细胞、EG 细胞,经形态学观察、AKP 染色、SSEA、OCT4染色、体外分化实验等,证明其具有胚胎干细胞的诸多特性。更多还原

【Abstract】 This test select goat to study the factor of effects on isolating and cloning Escells. This will develop establishing of goat ES and goat EG lone. The following is result: 1. Primodial germ cells were isolated and cultured from gonads or genital ridges of 46 fetus with age of 20~55days of gestation. Goat EG cell culture medium contains DMEM (high glucose), 15% NBS (or FBS), 0.1 mM ?-mercaptoethanol, 0.01 mM non-essential amino acid, 2 mM glutamine, 100 IU/ml penicillin, and 100 IU/ml streptomycin. One EG cell line had maintained an undifferentiated state for 6 passages. 30～36 days of gestation embryos are optimum for the isolation and culture of goat EG cells. It is difficult to isolate and culture goat EG cells if the age of fetuses is more than 6weeks of gestation. 2. Goat sertoli cells were isolated from goat testicle. Contracted PGCs growing on feeder of GSCs、MEF、GEF,it was found that GSCs>MEF>GEF. The DMEM supplement with 10% LIF is better on cloning goat EG cells. 3. 83 outgrowthing ICMs were obtained from 192 embryos of goat using whole method. ICMs were plated on mitomicin-inactivated MEF feeders and cultured in a humidified environment of 5% CO2 in air, 37 ℃. ES-like colonies were observed and one ES-like cell line had maintained undifferentiation for 8 passages.using 0.125% trypsin+0.02EDTA to digest. The method used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mM ?-mercaptoethanol, 0.01 mM non-essential amino acid, 10 ng/ml LIF, 100 IU/ml penicillin, and 100 IU/ml streptomycin. 4. There is no obvious different rate of attaching between the two feeders of MEF and GEF.(P>0.05) . they can promore growth of ES cells in vitro.with the development of goat ,the rate of attaching and multilplication was increasing. The rate of attaching and multiplication of hatched blastocyts is the highest, it was 90%and 60% respectly.the DMEM supplemented with 10% LIF is better on cloning goat ES cells. 5. Goat ES cells were not sensitive to trypsin, using 0.125% trypsin+0.02EDTA and”sereral times dissociation method”was the better way ,bescause this was relative gentle to ES cells and is better for forming new ES clones. 6. The goat ES and EG cells which were pluipotential cells have been indentified by colony morphology, AKP、SSEA、OCT4 staining, in vitrodifferentiation and karyotryping.更多还原