| 骆玉梅1,2 范勇1 陈欣洁1 李少英1 郑育红1 徐霞2△ 孙筱放1△.提高人胚胎干细胞建系率的优选培养体系与方法[J].,2012,12(11):2167-2172 |
| 提高人胚胎干细胞建系率的优选培养体系与方法 |
| The Modified Culture Medium and Special Method to Increase the Derivationof Human Embryonic Stem Cells |
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| DOI： |
| 中文关键词: 优选培养液 机械切割法 建系 人胚胎干细胞 |
| 英文关键词: Modified culture medium Mechanical cutting method Derivation Human embryonic stem cells |
| 基金项目:国家自然科学基金项目(30871378，31171229 )，广东省科技计划项目(2008B090500258、2009A030200010)和
广东省重大科技专项(20110812)资助 |
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| 中文摘要: |
| 目的：建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系，寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方
法，提高人胚胎干细胞(human embryonic stem cell, hESC)建系效率，建立人胚胎干细胞库。方法：将179 枚IVFDay3 废弃的胚胎放
入优选培养体系中培养(G2.5 培养液中添加10%人血清蛋白，人白细胞抑制生长因子(hLIF)，碱性成纤维细胞生长因子(bFGF))。
到Day7 将形成的囊胚全部用机械法分离ICM，接种于丝裂霉素C 灭活处理的原代小鼠胚胎成纤维细胞(MEF)上，培养8-9 天，每
4-5 天传代1 次。结果：优选培养体系的囊胚形成率为29.1%(52/179)，其中A 级囊胚形成率为11.2%(20/179)，50 个ICM 贴壁生
长，20 个出现克隆形态，成功建立11 株hESC(FY-hES-11 至FY-hES-21)。11 株hESC 均具有共同的多能性生物学特性。结论：优
选培养体系可以明显提高囊胚形成的质量，促进ICM 的增值，纯熟的机械切割法可以避免损伤ICM 并提高其贴壁率，原代灭活
的MEF 饲养层可以明显促进细胞增殖。 |
| 英文摘要: |
| Objective: To establish a modified culture medium to improve the blastocyst formation rate and quality from the discarded
embryos, and find good methods to promote the number of inner cell mass (ICM) increase, adherence and growth, so as to increase the
derivation of human embryonic stem cells (human embryonic stem cell, hESC ) and build hESC bank. Methods: We collected 179 Day3
discarded embryos into the modified culture medium (G2.5 medium supplemented with 10% human serum albumin, human leukocyte inhibiting
growth factor (hLIF), basic fibroblast growth factor (bFGF)). At Day7 separated ICM from the blastocyst and seeded on the
mouse embryonic fibroblasts (MEF) of passage 0 which have been inactivated by mitomycin C. The cells were cultured 8 to 9 days with
hESC medium and passaged every 4 to 5 days. Results: The data showed that the formation of blastocyst in the modified culture medium
was 29.1% (52/179), and the blastocyst formation rate with grate A was 11.2% (20/179). The number of ICM adherence were 50, and 20
clones appeared, finally established of 11 hESC which name were (FY-hES-11 to FY-hES-21). All of the hESC lines with common
pluripotent biological characteristic. Conclusions: Our Studies have shown that the modified culture medium can improve the blastocyst
formation rate and increase the number of the ICM. The proficiency skill of mechanical cutting method can avoid damage the ICM and
improve the rate of ICM adherence. MEF of passage 0 inactivated as feeder layer can promote cell proliferation significantly. |
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