传代培养对供核细胞胰岛素样生长因子Ⅱ基因表达的影响

Effect of Serial Subcultivation on the Expression of Insulin-like Growth FactorⅡ in Donor Cells

目的通过建立一种简便可靠的胰岛素样生长因子Ⅱ(IGF2)基因实时荧光定量PCR检测方法来探讨传代培养对供核细胞中IGF2基因表达的影响。方法通过实时荧光PCR,选择合适的"相对标准品"绘制相对标准曲线,检测来源于鼠耳的成纤维样细胞IGF2的表达。结果通过实时PCR的相对定量检测出在鼠耳成纤维样细胞的传代培养中,随着培养代数的增加,IGF2的表达减弱。结论双标准曲线的实时荧光PCR相对定量法用于检测IGF2的表达是可行的;培养代数影响IGF2基因的表达,为体细胞克隆研究中供核细胞的选择及后续的基因印迹研究奠定基础。

Objective To establish a reliable method of real-time PCR to detect the expression of imprinted gene IGF2 in serial subcultivated fibroblast-like cells from mouse ear. Methods Establishing the method of real-time PCR by selecting the suitable ＂relative standard preparation＂ and drawing the relative standard curve,which is used to detect expressive level of IGF2 in serial subcuhirated fibroblast-like cells. Results By the establishment of the method of real-time PCR,we detect that the expressive level of IGF2 decreases in the process of serial subcuhivation. Conclusion It is feasible to detect the expression of IGF2 by the method of real-time PCR of double standard curve. Serial subcuhivation effect the expressive level of IGF2 in fibroblast-like ceils from mouse ear, which provide bases for selection of donor cell and later study on imprinting in somatic cell nuclear transfer.