apelin对葡萄糖毒性作用与胰岛细胞pdx1蛋白表达的影响

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1、万方数据INFLUENCEOFAPELINONGLUCOSETOXICITYANDISLETCELLSEXPRESSIONOFPDX．1PROTEINABSTRACTobjective：1．ExploringwhetherApelinleadstothereductionofinsulinbyparticipatinginglucosetoxicitymechanism．2．StudyontherelationshipbetweenApelinandPDX一1proteinsynthesis．Method：NIT—

2、lcellsof1sletpcellstrainafterresuscitationandcultureiSinoculatedin25mlcellculturebottle(1xlO7cells／bottle)，culturingfor3daysinRPMI1640culturemedia，whichisdividedintosixgroupsrandomlybydifferentconcentrationofglucose：(1)GroupCon1，theconcentrationof5．6mmol／LGlu．

3、(2)GroupCon2，theconcentrationof5．6mmol／LGlu+Apelin一36(1lamol／L)．(3)GroupHG1，theconcentrationof16．7mmol／LGlu．(4)GroupHG2，theconcentrationof16．7mmol／LGlu+Apelin一36(1Irtmol／L)．(5)GroupEHG1，theconcentrationof33．3mmol／LGlu．(6)GroupEHG2，theconcentrationof33．3mmol／LG

4、lu+Apelin-36(1pmol／L)．Then：1．BasereleaseexperimentofIns：collect1mlcentrifugetube(1×100cells／tube)ofisletcellofeachgrouprespectively,andthenpre-incubatetheminHBSSfluidatthedegreeof37for60minutes，andafterPBSwashingfor2times，pre-incubatetheminHBSSbuffercontaining

5、Gluat5．6mmol／Latthedegreeof37for1hour,thendiscardthebufferandincubateeachcellrespectivelyinnew1mlHBSSbuffer一5．万方数据containingGlu(base)at5．6mmol／Landglucose(glucosestimulation)at16．7mmol／L，thencollectthereactionbufferandmeasuretheINSwiththemethodofELISA．2．INSinc

6、ell：collect1mlcentrifugetube(1xl06cells／tube)ofisletcellofeachgrouprespectively,afterfreezingandthawingrepeatedlyandextractingwiththeacidifiedalcohol，incubateatthedegreeof4。Cfor16—24hours，andthenmeasuretheINSwiththemethodofELISA．3．ExpressionofPDX一1protein：fixt

7、hesmearsoftheNITcellsfromthefourgroupswith4％paraformaldehydefor20minutesandthenPBSwashthemfor3minutesonetimefor3times，thenfollowtheinstructionoftheHistostain-PlusKit．Countthenumberofpositivecellswiththemicroscope．4．ExpressionofPDX-lmRNA：fixthesmearsoftheNIT-1c

8、ellsfromthefourgroupswith4％paraformaldehyde／0．1％DEPC／0．1mmol／LPBS(pH7．2～7．6、for20-30minutesandthenPBSwashthemfor3minutesonetimefor3times，thenfollowtheinstructionoftheInsituhybridiz