蛋白激酶C亚型在小鼠胚胎干细胞中的表达研究

Study on the Expressions of Protein Kinase C Isoenzyme in EmbryonicStem Cells of Mouse

[目的]研究5种蛋白激酶C(PKC)亚型PKC_α、PKC_β、PKC_γ、PKC_δ和PKC_ε在小鼠胚胎干细胞(ES细胞)株ES-BALB/c的表达情况。[方法]ES-BALB/c细胞株用普通ES细胞培养液培养,分别种植于预置有盖玻片的6孔板和直径100mm的培养皿,培养2d和4d后分别进行PKC亚型免疫组织化学和Western印迹检测。[结果]免疫组化发现PKC_α在ES-BALB/c细胞有广泛的棕黄色的阳性表达,以细胞浆和细胞核最甚,而PKC_β、PKC_γ、PKC_δ和PKC_ε4种亚型无明显表达;Western印迹发现PKC_α出现一条蛋白印迹条带,分子质量约为84ku,而PKC_β、PKC_γ、PKC_δ和PKC_ε4种亚型无蛋白印迹条带。[结论]PKC_α在ES细胞阶段即有表达,在将来的细胞分化阶段可能有非常重要的作用,而PKC_β、PKC_γ、PKC_δ和PKC_ε4种亚型在ES细胞阶段没有表达。

[Objective] To study the expressions of the protein kinase C(PKC) isoenzyme PKCα, PKCβ, PKCγ, PKCδ and PKCε in embryonic stem cells(ES cells) of mouse. [Methods] ES-BALB/c cells were incubated in the ES conditioned medium, and cultured in 6-well culture dishes with coated glass coverslip and in 100-mm dishes for 2 and 4 days, respectively. Immunohistochemistry and Western blot assay were performed to identify the PKC isoform expression. [Results] Staining of PKCα but PKCβ, PKCγ, PKCδ and PKCε was strong and distributed throughout the cells, especially in the cytoplasm and nucleus. Western blot analysis exhibited a band of approximately 84 ku with the antibody specific to PKCα. No reactions were shown for other four PKC isoenzyme, PKCβ, PKCγ, PKCδ and PKCε. [Conclusion] PKCα is expressed at the stage of ES cells and probably plays an very important role in differentiation , while PKCβ,PKCγ, PKCδ and PKCε are not expressed at the same time.