【作者】 洪俊君；

【导师】 王锋；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 本试验以小鼠ES-D3细胞为材料，对ES-D3细胞的饲养层体系、冷冻保存体系以及质粒pNl-EGFP瞬时转染体系分别进行优化，以期找到最佳的培养、冻存和转染体系。为后期开展胚胎干细胞（ES）介导的转基因动物生产途径提供研究基础．本试验的具体试验內容如下：1．试验一以昆明白小鼠13.5d的胚胎为原料，分别用三种不同的消化方法对胚胎组织进行消化以获得原代小鼠胚胎成纤维细胞（PMEF）。同时研究了不同温度、丝裂霉素C处理时间长短以及支原体污染与否对MEF生长性能的影响。试验结果表明，不同分离方法对PMEF的成活率与贴壁率有显著影响，当用5～10ml 0.25％胰蛋白酶-0.04％EDTA.2Na，在37℃、5％CO₂、饱和湿度培养箱中孵育消化小鼠胚胎组织5～10min时获得的PMEF成活率（91.83±3.17％）和贴壁率（87.50±1.22％）显著高于其他三个试验组。获得的小鼠胚胎成纤维细胞形态特征典型，细胞透明，胞质向外伸出伪足，形成梭形，少量呈不规則的三角形、星形，细胞轮廓清晰，间隙明显．试验发现，MEF对温度敏感，37℃状态下细胞贴壁率（87.50±1.29％）最高，生长状态最佳．随着温度的升高，细胞贴壁率明显下降，37.8℃时，细胞贴壁率不到30％。用10μg／mL的丝裂霉素C处理MEF制作ES细胞的饲养层时，最佳处理时间为2.5h．丝裂霉素处理时间对饲养层的优劣具有很大影响．处理时间太短无法抑制饲养层细胞的生长，而处理时间过长又会引起饲养层细胞的死亡，从而影响ES细胞的生长。对MEF细胞进行支原体污染检测发现，支原体污染对MEF的生长性能有显著影响，受污染的胚胎成纤维细胞形态不典型，细胞凋亡现象严重，无法用作ES细胞的饲养层．2．试验二以小鼠ES-D3细胞系为试验材料，对比分析了不同血清来源和血清浓度对ES细胞冻存效率的影响；同时研究了不同转染方法对ES-D3细胞外源基因（pNl-EGFP）转染效率的影响．试验结果表明，不同血清浓度对ES-D3细胞的冻存效率有显著影响。ES-D3细胞在冻存液血清浓度为60％时，细胞解冻活率佳（与90％血清浓度下细胞的解冻活率无显著差异），且细胞克隆形状典型，AKP检测呈阳性，在体外悬浮培养状态下能形成典型的EB状组织．不同血清来源，国产胎牛血清和进??口胎牛血清，对ES-D3细胞的冻存效率有显著影响。对ES-D3细胞转染外源基因pS1-EGFP时，悬浮转染方法能获得88.72a：0.91％的转染效率，但是转染后ES-D3细胞克隆形态不典型，易发生分化。贴壁转染时，在优化条件下（DNA∶Lipofeetamine2000=2μg∶12μl）ES-D3的转染效率可以达到74.45±2.16％，转染效率显著低于悬浮转染效率，但转染后ES-D3细胞克隆形态典型，分化现象不明显。更多还原

【Abstract】 In this study, mouse ES-D3 cells were used for optimizing ES-D3 cells feeder cellculture system, cryopreservation system and the plasmid pN1-EGFP transient transfectionsystem, in order to find the best culturing, cryopreservation and transfection system, andprovide bases for the later research on production of transgenic animals mediated byembryonic stem cells （ES）. The specific test results were as follows:1. In this paper, mouse 13.5d old embryos were used to be digested by three differentmethods to obtain primary mouse embryonic fibroblasts （PMEF）, and the effect of differenttemperature, time with mitomycin treatment, myeoplasma contamination on the MEFgrowth performance were investigated..The results showed that effects on cell survival rateand adherent rate of different digestion methods were significant When digested mouseembryos with a 5～10ml 0.25％trypsin-0.04％EDTA. 2Na, at 37℃, 5％CO₂, saturatedhumidity incubator for 5～10 rain, cell survival rate （91.83±3.17％） and adherent rate（87.50±1.22％） were significantly higher than the other three groups. The mouse embryonicfibroblasts had typical features. The results showed that the MEF were temperaturesensitive. Under 37℃, cell adhesion rate （87.50±1.29％） was the highest, which indicated37℃was the best growth temperature. With the increase of temperature, adherent cells ratewas significantly decreased. At 37.8℃, cell adhesion rate was less than 30％. When treatedMEF with 10μg/mL mitomycin C to produce feeder cells, the optimal treatment time was2.5h. Time with Mitomycin C treatment have a great influence on the feeder ceils. Iftreatment time is too short, the cells growth can’t be inhibited, or if the processing time istoo long, it will cause the death of the feeder cells, thus affecting the cultivation of ES cells.MEF cells of mycoplasma contamination were also detected, mycoplasma contaminationhad a significant effect on the growth performance of MEF, contaminated embryonicfibroblasts morphology were not typical, and apoptosis was severe. 2. ES cells cryopreservation efficiency were analysed with different varieties andconcentration of serum, and different transfection methods with the exogenous gene（pN1-EGFP） were also used to study the transfection efficiency on ES-D3 cells. The resultsshowed that the effect of different serum concentration on frozen efficiency of ES-D3 cellswas siguificant. When serum concentration in the frozen liquid was 60％, the rate of livecells after thawing was the best with typical cell clones shape and positive AKP. Undersuspension culture in vitro, the cells could be a typical EB-like organizations. Withdifferent serum varieties of domestic and imported fetal calf serum （FCS）, the ES-D3 cellsfrozen efficiency was significantly different. When the ES-D3 cells were transfected withexogenous gene pN1-EGFP, the transfection efficiency of suspended transfection methodwas 88.72±0.91％. But alter transfection, ES-D3 clones forms were not typical, and easy todifferentiate. Adherent transfection, under the optimum conditions （DNA:Lipofectamine2000=2μg: 12μL）, ES-D3 transfection efficiency could be achieved at74.45±2.16％. Transfection efficiency was significantly lower than suspension transfectionefficiency, but ES-D3 clones had typical morphology and differentiation phenomenon wasnot apparent alter transfection.更多还原