ICR小鼠胚胎干细胞建系初步研究

实验旨在探讨消化方式和胚胎发育阶段对ICR小鼠胚胎干细胞(ES细胞)建系效率的影响。ICR小鼠3.5 d囊胚在饲养层上贴壁后采用单一酶消化或机械化与酶消化法相结合分离隆起的细胞集落,进行传代培养;然后选择二者中较优消化方式对不同发育时期囊胚所形成的细胞集落进行处理。结果表明:采用机械化与胰酶消化相结合的方式,形成的类ES细胞超过7代的比率(85.0%)要显著高于单一的胰酶消化(15.0%)(P<0.05);当用二者相结合的方式对ICR小鼠3.5 d(早期囊胚)、4.0 d(扩张囊胚)和4.5 d(孵化囊胚)所形成的细胞集落进行消化传代培养,三者在贴壁率和形成原代细胞集落率上均无显著差别(P>0.05),但传代超过7代的效率上早期囊胚和扩张囊胚均高于孵化囊胚(P<0.05)。结果提示,采用机械化与酶消化法相结合更适合于3.5～4.0 d ICR小鼠囊胚的ES细胞建系。

Establishment of Embryonic Stem Cell Line Derived from ICR Mouse Blastocyst

To observe the effect of embryonic age and the type of disassociation on the efficiencies of ES cell derivation from ICR mouse blastocysts.Firstly,day 3.5 mouse blastocysts were cultured on mouse embryonic fibroblast(MEF) feeder layer for inner cell mass(ICM) formation,and the ES cell colonies from the ICM were mechanically dissected or disassociated by trypsin for passaging and subsequent propagation.Then the better method between them was selected to treat the cell colonies from day 3.5 to 4.5 blastocyst.Results showed that more than passage 7 ES cells clone rate(85.0%) from colonies dissceted mechanically and by trypsin were significantly higer(P<0.05) than that(15.0%) from the colonies dissected by trypsin alone.When the ES cell colonies from the day 3.5,4.0 and 4.5 mouse blastocysts were disassociated for passaging by the above better program,the attachment rate(100.0%,100.0% and 96.7%) and ICM formation rate(89.3%,88.9% and 90.0%) from day 3.5 to day 4.5 blastocysts were similar(P>.05).But the(more than) passage 7 ES cells clone rate from the day 3.5 to day 4.0 embryos(83.3 to 84.0%) were signicantly higher(P<0.05) than that(22.2%) from day 4.5 blastocysts.ES cell colonies dissected mechanically and by trypsin was suitable for the establishment of ES cell line from ICR mouse day 3.5 to 4.0 blastocysts.