| γ射线预处理制备胚胎干细胞滋养层的实验研究 |
| 投稿时间：2009-12-24 修订日期：2010-01-27 点此下载全文 |
| 引用本文：刘尧,杨明,王惠,杨卫兵,秦茂林,李红丽.γ射线预处理制备胚胎干细胞滋养层的实验研究[J].医学研究杂志,2010,39(4):33-37 |
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| 基金项目:广西壮族自治区科学研究与技术开发基金资助（桂科攻0592007-1G）；解放军第一八一医院科研基金 |
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| 中文摘要:目的筛选并建立稳定、高效的胚胎干细胞滋养层细胞体外培养的最佳条件，利于胚胎干细胞的培养。方法清洁级昆明系孕12.5天、14.5天、18.5天雌鼠和新生(P0)昆明小鼠各5只。无菌取上述各孕龄小鼠胚胎分离培养原代鼠胚成纤维细胞（MEF）并传代；以60Coγ射线（30Gy，20℃）分别照射10、20、25min后制备滋养层细胞。结果胚龄及60Coγ射线照射对MEF的细胞活性、生长增生和分泌功能有影响。（1）细胞活性状态：①E12.5天、E14.5天、E18天鼠胚和P0来源的第3~5代MEF活性均较好。E18.5天鼠胚和P0来源的第6代MEF，后期深染变形细胞的比例增多达32%，与E14.5天鼠胚来源同代细胞相比有显著差异（P<0.05）；②E14.5天来源的第3~6代细胞采用60Coγ射线照射10min组，细胞仍生长迅速。照射20min组，细胞可维持存活达20天以上，细胞密度无显著变化。照射25min组，细胞存活14天左右且细胞密度无显著增加。（2）细胞分泌功能：①E14.5天鼠胚来源的第3~6代MEF以及用60Coγ射线照射10min组和20min组并培养5天时均可见较多数目的Ⅰ型胶原蛋白免疫阳性细胞，且染色较深；②60Coγ射线照射25min组，培养5天后第3~4代MEF中Ⅰ型胶原蛋白阳性细胞数目与正常组相比无明显差异；但第5代后MEF中Ⅰ型胶原蛋白阳性细胞数目减少，染色浅淡。结论E14.5天鼠胚来源的第3~6代是用于制备滋养层的最佳来源；60Coγ射线（30Gy，20℃）照射20min可作为预处理制备滋养层细胞的最佳条件。 |
| 中文关键词:小鼠胚胎成纤维细胞（MEF） 胚胎干细胞（ESC） 滋养层 60Coγ 射线 |
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| Study on the Pretreatment of Embryonic Stem Cell Feeder Layers by γ-Irradiation |
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| Abstract:AbstractObjectiveTo establish a stable and effective system of embryonic stem cell feeder layer in order to culture embryonic stem cell in vitro. MethodsMouse primary embryonic fibroblasts were sterilely prepared from mouse fetus of different embryonic ages. When embryonic fibroblasts grew and contacted each other，they then were treated with 60Coγ-irradiation for 10, 20 and 25 minute. ResultsThe cell viability, proliferation and secretion were affected by the embryonic age and 60Coγ-irradiation. (1) Viability: ①All the passages of MEFs from third to fifth, derived from E12.5, E14.5, E18.5 embryon and P0 postnatal mouse, had good cytoactives.The sixth passage of MEFs from E18.5 and P0 mouse had more than 32% amoeboid cells and were significantly different from the same passage of E14.5 cells (P<0.05); ②The third to sixth passages of MEFs form E14.5 embryon still grew quickly after 10min irradiation, survived more than 20 days with remaining cell density after 20min irradiation, and survived about 14 days with no changes cell density after 25min irradiation. (2) Secretion: ① MEFs from third to sixth passage, derived from E14.5 embryon, after 60Coγ-irradiation and cultured for 5 days, were strongly I-type collagen protein positive cells; ②For MEFs from third to fourth passage, after 25min 60Coγ-irradiation and cultured for 5 days, the number of positive cells of I-type collagen protein was no different with control group; but after fifth passage, the positive cells of I-type collagen protein decreased and light dying.ConclusionThe optimal origin of feeder layers were the third to sixth passages.E14.5 derived MEFs 20min 60Coγ-irradiation (30 Gy, 20℃) was the optimal condition for preparing feeder layer cells. |
| keywords:Mouse embryonic fibroblast(MEF) Embryonic stem cell（ESC） Feeder layers 60Coγ-irradiation |
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