摘要
目的研究视黄醇在白血病抑制因子(LIF)维持小鼠胚胎干细胞(mESC)多潜能性中的协同作用。方法实验分为两组,在相同LIF浓度(100U/ml)的mESC常规培养基础上,实验组添加视黄醇,对照组则不添加视黄醇。培养14d后,观察两组碱性磷酸酶(ALP)染色情况及mESC的形态变化,通过RT-PCR及Western blot检测Oct4和Nanog的mRNA及蛋白表达情况。结果两组mESC常规培养14d后,实验组78%mESC处于未分化状态,细胞集落形态维持较好,ALP染色强阳性;而对照组中仅有35%mESC处于未分化状态,ALP染色弱阳性;实验组未分化细胞数量(7.02±0.34)明显高于对照组(4.28±0.37,P<0.05)。RT-PCR及Western blot结果显示:实验组Nanog及Oct4的mRNA表达(分别为204.95±11.24,160.69±11.52)均高于对照组(分别为122.89±9.25,88.21±4.69,P<0.05);实验组Nanog及Oct4的蛋白表达(分别为238.14±14.72、187.16±9.02)均明显高于对照组(分别为205.04±10.03,132.41±15.78,P<0.05)。结论在中等浓度LIF因子作用时添加视黄醇对维持mESC多潜能性有明显作用。
Objective To investigate the synergistic effect of retinol and leukemia inhibitory factor （LIF） on maintaining pluripotency of murine embryonic stem cells （mESCs）. Methods The mESCs were randomly divided into two groups, and they were all cultured with the same LIF concentration （100U/ml）. Retinol was added into mESCs medium in experimental group, and retinol was not added in control group. 14 days after culturing, the changes in cell morphology were observed under an inverted microscope with alkaline phosphatase staining. The mRNA expression of Oct4 and Nanog was analyzed by RT-PCR, and the proteins for Oct4 and Nanog were determined by Western-blot for the assessment of the pluripotency of mESCs. Results 14 days after culturing, 78% of mESCs in the experimental group formed good cell colonies with good stereoscopic appearance, and they showed strong alkaline phosphatase （AKP） staining. While in the control group, only 35% of mESCs were in undifferentiated mode, and the cells were weakly positive with AKP staining. The quantity of undifferentiated mESCs in experimental group （7.02±0.34） was higher than that in control group （4.28±0.37, P〈0.05）. RT-PCR results indicated that both the Nanog and Oct4 mRNA expressions were higher in experimental group （204.95±11.24, 160.69±11.52） than that in control group （122.89±9.25, 88.21±4.69; P〈0.05）. Western blotting indicated that Nanog and Oct-4 expressions were also higher in experimental group （238.14±14.72, 187.16±9.02） than that in control group （205.04±10.03, 132.41±15.78, P〈0.05）. Conclusion Retinol showed a synergistic effect in maintaining pluripotency of mESCs when combined with LIF in moderate concentration, and the effect may be attributable to the over-expression of Nanog under retinol stimulation.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第4期425-427,共3页
Medical Journal of Chinese People's Liberation Army
