【摘要】 烟碱样乙酰胆碱受体 (nAChR)的表达调控受神经电活动影响 ,电刺激引起肌细胞膜去极化可抑制nAChR的表达 .以往的研究表明 ,Ca2 +和PKC以及生肌素在其中发挥着重要的作用 .然而 ,目前尚不清楚究竟是哪种PKC亚型参与此过程 ,PKC激活对特异转录因子生肌素浆核转位有何影响 ?为探讨PKC在去极化 nAChR转录偶联中的作用 ,构建了含nAChRγ亚基启动子的绿色荧光蛋白 (GFP)表达载体pEGFP γ ,将其分别与 4种cPKC(PKCα、PKCβⅠ、PKCβⅡ、PKCγ)真核表达载体共转染C2C12肌细胞 .结果发现PKCβⅠ、PKCβⅡ对nAChRγ启动子驱动的GFP报告基因表达没有影响 (P >0 .0 5 ) ,PKCγ对报告基因表达有抑制作用 (P <0 .0 5 ) ,PKCα则有明显抑制作用 (P <0 .0 1) .采用 4种cPKC真核表达载体与GFP 生肌素融合蛋白表达载体 (pGFP myog)共转染C2C12肌细胞 ,观察了不同亚型PKC表达对生肌素浆至核转位的影响 ,发现只有强制性表达外源性PKCα可明显抑制生肌素向核中转位 ,而PKCβⅠ、PKCβⅡ及PKCγ对生肌素浆核转位没有明显抑制作用 .结果提示 ,PKCα通过抑制生肌素转位是阻遏nAChR基因表达机制之一 .更多还原

【Abstract】 It is well documented that electricity regulates the expression of nicotine acetylcholine receptor(nAChR).Electricity induced depolarization inhibits the expression of nAChR,in which calcium,protein kinase C and myogenin play major roles.Calcium elevation and activation of PKC inhibit the expression of nAChR.It is still uncertain which isoform of PKC participates in the inhibitory process and how protein kinase C regulates myogenin nuclear translocation.The current work is the basis for further exploration of mechanism of the coupling of depolarization with nAChR gene transcription.The pEGFP γ expression vector,in which GFP reporter gene expression was driven by the γ promoter of nAChR,was constructed.pEGFP γ was co transfected with four conventional PKC expression vectors,PKCα,PKCβⅠ,PKCβⅡ and PKCγ,respectively.PKCβⅠ and PKC βⅡ had no effects on the expression of pEGFP γ.PKCγ inhibited the expression of pEGFP γ( P < 0.05 ).PKCα greatly inhibited the expression of pEGFP γ( P <0.01).GFP myogenin fusion protein expression vector pEGFP myog was constructed and co transfected into C2C12 muscle cells with four conventional PKC expression vectors also.PKCβⅠ,PKCβⅡ and PKCγ had no impacts on GFP myogenin nuclear translocation,whereas PKC α could inhibit myogenin translocation from cytosol to nucleus.These results suggest that activated PKCα may inhibit myogenin translocation to the nucleus.Thus the transcription of nAChR γ subunit is inhibited.更多还原