Department of Histology and Embryology, the Second Military Medical University, 800 Xiangyin Road, Yangpu district, Shanghai 200433, China.
Reprod Biomed Online. 2012 Oct;25(4):425-33. doi: 10.1016/j.rbmo.2012.06.018. Epub 2012 Jul 8.
As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. The present study found that the use of cytochalasin B was not necessary for, and even harmful to, the enucleation of human zygotes. This study also decreased the DNA methylation levels in reconstructed embryos using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), in an attempt to correct the abnormalities in DNA methylation that might play an important role in the failure of embryo development. After 5-aza-dC treatment and nuclear transfer (NT-Aza group), 32.7% of reconstructed embryos developed to the 8-cell stage, which is a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that of the NT group, as determined by 5-methylcytosine immunodetection. Based on the present results, this study recommends performing the enucleation procedure without cytochalasin B treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies.
作为一种涉及较少伦理问题的丰富来源，人类异常受精的胚胎在核转移治疗性克隆中作为核受体比卵母细胞更具优势。然而，应该开发更有效的操作条件，用于使用人类异常受精的胚胎作为核受体的体细胞核转移（SCNT）研究。本研究发现，对于人胚胎的去核，细胞松弛素 B 的使用不是必需的，甚至是有害的。本研究还使用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷（5-aza-dC）降低了重构胚胎中的 DNA 甲基化水平，试图纠正可能在胚胎发育失败中起重要作用的 DNA 甲基化异常。经过 5-aza-dC 处理和核转移（NT-Aza 组）后，32.7%的重构胚胎发育到 8 细胞期，这一比例明显高于仅核转移（NT）组（11.1%）。通过 5-甲基胞嘧啶免疫检测发现，NT-Aza 组的 DNA 甲基化水平明显低于 NT 组。基于目前的结果，本研究建议在人类 SCNT 研究中进行核受体的去核操作时不使用细胞松弛素 B 处理，并在重构胚胎的培养中使用 5-aza-dC。
