激活wnt通路促进nit-1胰岛β细胞pparγ表达的研究-study on activation of wnt pathway to promote ppar γ expression in nit - 1 islet β cells.docx

激活Wnt通路促进NIT-1胰岛β细胞PPARγ表达的研究硕士研究生：周博文导师：袁刚教授中文摘要目的：通过体外培养干预小鼠NIT-1胰岛β细胞，观察Wnt信号通路对过氧化物酶体增生物激活受体γ（peroxisomeproliferator-activatedreceptorγ，PPARγ）及葡萄糖激酶（glucokinase，GK）表达的调节作用，并探讨Wnt信号通路与PPARγ在β细胞中的复杂对话。方法：重组Wnt3a蛋白干预体外培养的小鼠NIT-1胰岛β细胞，荧光定量PCR及WesternBlot方法检测PPARγmRNA及蛋白水平较对照组的变化，并给予Wnt通路阻断剂dickkopf1（DKK1）及PI3K/Akt通路阻断剂wortmannin（Wort），比较PPARγ的表达量的改变。PCR法检测各干预组（对照组、Wnt3a组、Wnt3a+DKK1组、Wnt3a+Wort组）GKmRNA的表达。结果：细胞Wnt信号通路激活，PPARγmRNA水平较对照组增加了41.2%±7.8%（p0.05），蛋白表达较对照组增加了97.8%±29%（p0.05），GK的mRNA水平较对照组增加了65%±14.8%（p0.05）。DKK1阻断Wnt信号通路后，明显抑制了PPARγ与GK的mRNA表达，PPARγ的蛋白水平亦较Wnt3a干预组降低了36.2%±7.7%（p0.05），激活Wnt通路同时以Wort阻断PI3K通路，发现PPARγ与GK的mRNA水平下降，PPARγ的蛋白水平亦较单纯Wnt3a干预组降低了28.1%±3.8%（p0.05）。同时阻断Wnt及PI3K通路时，发现PPARγ的蛋白水平下降的更为显著，较Wnt3a组降低83.4%±4.9%（p0.05），作用较单阻断Wnt或PI3K信号通路时更明显（p0.05）。结论：1.在小鼠NIT-1胰岛β细胞，激活Wnt信号通路，能够上调PPARγ及GK的表达，影响细胞胰岛素分泌功能；2.Wnt3a对NIT-1细胞PPARγ的刺激作用部分依赖于PI3K/Akt通路的作用。关键词：WntPI3KPPARγβ-cateninTCF7L2AktGKStudiesontheEffectsofPPARγInducedbyWntSignalingPathwayinNIT-1βCellPostgraduate:ZhouBowenTutor:Prof.YuanGangAbstractObjective:Tostudytheeffectofperoxisomeproliferator-activatedreceptorγ(PPARγ)andglucokinase(GK)inducedbyWntsignalingpathwayinmiceNIT-1pancreaticβcell,andtoexploretheinteractionalrolesbetweenPPARγandWntsignalingpathways.Methods:WntsignalingpathwaycanbeactivatedbyrecombinatedWnt3aproteinandblockedbydickkopf1(DKK1),andphosphatidylinositol3kinase(PI3K)signalingpathwaycanbeblockedbywortmannin(Wort)inNIT-1cells.Cellsweredividedintofourgroupsaccordingtodifferentinterventions:controlgroup,Wnt3agroup,Wnt3a+DKK1groupandWnt3a+Wortgroup.After24hourcultivation,mRNAlevelsofPPARγandGKweredetectedbyfluorescentquantitationofrealtimePCR.WesternBlotwasusedtocomparetheproteinlevelsofPPARγamongthedifferentgroups.Results:ThemRNAandproteinlevelsofPPARγwereelevatedinducedbytheactivationofWntpathway,andexpressionofGKgenealsobestimulated.TheseeffectswereabrogatedbyinhibitionsofWntor(and)PI3K,DKK1or(and)Wort.Conclusions:TheseresultsindentifiedthatPPARγandGKcanbeup-regulatedbyWntsingnal