亲磷脂酸磷脂酶A1基因沉默降低MIN6细胞胰岛素分泌水平.pdf

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1、2872实用医学杂志2014年第30卷第18期·基础研究·亲磷脂酸磷脂酶A1基因沉默降低MIN6细胞胰岛素分泌水平莫之婧苏何玲朱华李红岩杨易史云龙黄海霞刘永明摘要目的：探讨亲磷脂酸磷脂酶A1(PA．PLA1)基因沉默对小鼠分泌胰岛素细胞MIN6分泌功能的影响方法：根据GenBank中小鼠PA—PLA1基因mRNA序列构建siRNA表达载体(pGPU6PA．PLA1)并转染MIN6．实时荧光定量PCR和Westernblot筛选有效干扰载体。将获得的有效干扰载体转染MIN6细胞48h后进行葡萄糖刺激实验，检测其胰岛素分泌的

2、变化。结果：酶切及测序证实成功构建了4个靶向PA—PLA1的siRNA表达栽体。实时荧光定量PCR和Westernblot分析显示siRNA表达载体pGPU6一PA—PLA1．1885具有较高的干扰效率．其转染的MIN6细胞PA．PLA1mRNA水平降至对照组的46．3％，PA—PLA1蛋白水平降至对照组的33．9％。同时胰岛素分泌水平降至对照组的65．0％(P<0．05)。结论：PA．PIA1基因沉默可降低MIN6的胰岛素分泌水平。关键词亲磷脂酸磷脂酶A1：siRNA；基因沉默；胰岛素分泌Genesilencingof

3、phosphatidicacid-preferringphospholipaseA1decreasesinsulinsecretioninMIN6cellsMOZhi-jing，SUHe—ling，ZHUHua，LIHong-yan，YANGYi，SHIYun·long，HUANGHai-xia，LIUYong-ming．KeyLaboratoryofMedicinalBiotechnologyandDepartmentofBiochemistryandMolecularBiology，GuilinMedicalUniv

4、ersity，Guilin541004，ChinaCorrespondingauthor：LIUYong-mingE—mail：liuym@glmc．edu．cn【Abstract】objectiveToexploretheeffectofthegenesilencingofphosphatidicacid-preferringphospholipaseA1(PA—PLA1)oninsulinsecretioninmouseinsulin—secretingcelllineMIN6．MethodsThesiRNAexpr

5、essionvectorofmousePA-PLA1genetargetingwasconstructedusingmousePA—-PLA1mRNAsequenceavailableinGenBank，andMIN6cellsweretransfectedwiththevector．FluorescencequantitativePCRandWestern·blotwereappliedtoscreenefficientRNAi—vector．AftertransfectionwithobtainedeficientR

6、NAi—vectorsfor48hours，glucose—stimulatedinsulinsecretionexperimentswereconducted，andthechangesofinsulinsecretionwereexamined．ResultsFoursiRNAexpressionvectorsofnlonsePA·PLA1genetargetingwereconfirmedtobesuccessfullyconstructedbytheanalysesofenzymecleavageandseque

7、ncing．TheresultsoffluorescencequantitativePCRandWesternblotanalysesindicatedthatthesiRNAexpressionveetorpGPU6一PA—PLAI一1885wasthemosteffectiveRNAi—vectorinthefourvectors．TheexpressionlevelsofthePA—PLA1mRNAandproteinoftheMIN6cellstransfectedwithpGPU6一PA—PLA1—1885de

8、creasedto46-3％and33．9％ofthatofthecontrol，respectively，andmeanwhiletheinsulinsecretionlevelsofthecellsdecreasedto65．0％ofthatofthecontrol(P