Shi C Z, Dhir R N, Kesavan P, Zhang S L, Matschinsky F M, Heyner S
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104-6080, USA.
Mol Reprod Dev. 1995 Oct;42(2):173-9. doi: 10.1002/mrd.1080420206.
Insulin and insulin-like growth factors (IGF-I and -II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT-PCR), and at the protein level by radioactive labeled ligand-receptor binding assay. Using RT-PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42 degrees C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF-I, or IGF-II receptors by PCR. RT-PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size.(ABSTRACT TRUNCATED AT 250 WORDS)
胰岛素及胰岛素样生长因子（IGF-I和IGF-II）是一类生长因子家族的成员，已知在小鼠植入前胚胎发育过程中受到发育调控。胰岛素家族生长因子的生理作用是通过与特定细胞表面受体相互作用介导的，这些受体在小鼠植入前胚胎细胞上可检测到。小鼠胚胎干细胞（ES细胞）是直接从囊胚内细胞团衍生而来的全能细胞。ES细胞有能力分化为所有三个胚层，并且在特定培养条件下具有无限的生长潜力。与植入前胚胎相比，ES细胞的一大优势是能够获得大量用于生化研究的组织。为了更详细地研究胰岛素家族生长因子的生物学作用，检测了它们在ES细胞上同源受体的表达。ES细胞在添加白血病抑制因子（LIF）的DMEM培养基中培养以维持未分化状态。使用逆转录聚合酶链反应（RT-PCR）在mRNA水平评估受体表达，并通过放射性标记配体-受体结合试验在蛋白质水平进行评估。使用RT-PCR在ES细胞中检测到了所有三种生长因子受体的mRNA。ES细胞的信使RNA在42℃下用禽成髓细胞瘤病毒逆转录酶（AMV逆转录酶）逆转录为cDNA 1小时。通过PCR用Taq聚合酶和胰岛素、IGF-I或IGF-II受体的特异性引物扩增逆转录反应。RT-PCR和对照质粒cDNA PCR产物在3%琼脂糖凝胶上进行电泳分离。每个扩增的PCR产物都显示出预测的正确大小。（摘要截断于250字）
