TrkB-BDNF信號傳導(dǎo)通路對神經(jīng)母細胞瘤細胞耐藥和轉(zhuǎn)移的影響.pdf

1、TrlcB—BDNF信號傳導(dǎo)通路對神經(jīng)母細胞瘤細胞耐藥和轉(zhuǎn)移的影響摘要目的TrKB～BDNF信號傳導(dǎo)通路與神經(jīng)母細胞瘤(Neuroblastoma，NB)細胞的化療耐藥和轉(zhuǎn)移密切相關(guān)。本研究探討阻斷TrkBBDNF信號傳導(dǎo)通路的三條下游信號傳導(dǎo)通路后，NB細胞SHSY5Y對化療藥物順鉑的敏感性及分泌基質(zhì)金屬蛋白酶一9(matrixmetalloproteinase一9，UP一9)的影響。方法常規(guī)培養(yǎng)SH—SY5YNB細胞，用納摩爾濃度全

2、反式維甲酸(A1ltransRetinoicAcid，ATRA)誘導(dǎo)酪氨酸激酶受體B(TyrosinekinasereceptorB，TrkB)高表達，加入腦源性神經(jīng)營養(yǎng)因子(Brain—derivedneurotrophicfactor，BDNF)，從而激活TrkBBDNF信號傳導(dǎo)通路。用磷脂酰肌醇一3激酶(P13K)抑制劑LY294002、磷脂酶C(PLC)抑制劑U73122、絲裂原活化蛋白激酶(MAPK)抑制劑U0126阻斷該信號

3、通路的相應(yīng)下游信號傳導(dǎo)通路后，加入化療藥順鉑(CDDP)。四甲基偶氮藍比色(MTT)實驗方法檢測應(yīng)用三種抑制荊前后細胞的存活率的變化；流式細胞儀(FCM)檢測應(yīng)用三種抑制劑前后細胞凋亡率的變化。酶聯(lián)免疫吸附測定(ELISA)技術(shù)檢測全反式維甲酸(ATRA)誘導(dǎo)下酪氨酸激酶受體B(TrkB)、腦源性神經(jīng)營養(yǎng)因子(BDNF)水平SH—SY5Y細胞培養(yǎng)上清中MMP一9含量及應(yīng)用三種抑制劑前后SHSYSY細胞培養(yǎng)上清中MMP一9含量的變化。結(jié)果

4、ATRABDNFCDDP組NB細胞的存活率及凋亡率與對照組相比，差異無顯著性(PO05)。ATRABDNFLY294002CDDP組細胞對順鉑的敏感性增加，細胞的存活率明顯降低，凋亡率明顯升高，與ATRABDNFCDDP組相比，差異有顯著性(PO05)；50ng／ml、IOOng／mlBDNF組mlP9含量均明顯高于對照組(p005)；ATRABDNF組姍P一9EffectsoftheTrKB—BDNFsignalpathwayoRth

5、echemoresistaneeandmetastasisofneuroblastomacellsAbstractObjectiveTrkB—BDNFsignalpathwayishighlyassociated謝tllthechemoresistanceandmetastasisofneuroblastoma(NB)cellsInthisstudywedescribedthechangesofthesensibilityofNBSHS

6、Y5Ycellstochemotherapydrugcisplatin(CDDP)andthesynthesisandsecretionofmatrixmetalloproteinases一9(MMP一9、beforeandafterblockageofl礬圓一BDNFdownstreamsignatpathwaysMethodsHamanNBcelllineSHSY5Y(SYSY)wasroutinelyculturedHighexp

7、ressionoftyrosinekinasereceptorB(TrkB)wasinducedbynMall—transretinoidacid(ATRA)Thenaddtheectogenidbrain—derivedneurotrophlcfactor(BDNF)toactivatetheTrld3一BDNFsignalpathwayThethreedownstreamsignalpathwaywasrespectivelyinh

8、ibitedbyLY294002(theP13Kpathwayinhibitor)、U73122(thePLCpathwayinhibitor)、U0126(theMAPKpathwayinhibitor)Cell_viabilityWaSassessedbymethylthiazolyltetrazolium(MTT)assayCellapoptosisratewasdetectedbyfloweytometry(FCM)MMP一9c

9、oncentrationsintheSY5Ycellculturesupematantswere‘measuredbyenzyme—linkedimmunosorbentassay(ELISA)afterthetreatmentofdifferentlevelsof1拙andBDNFinducedbyATRAThechangeof^廈^—口9concentrationsintheSY5Ycellculturesupematantswer

10、emeasuredbyELISAbeforeandafterthethreepathwayinhibitorswereusedResultsTheviabilityandapoptosisrateoftheSY5YcellstreatedwithATRA，BDNFandCDDPwerenotdifferentfromtheblankcontrol口oup@O05)HoweverthesensibilityofSY5Ycellstoche

11、motherapydrugCDDPincreasedinthegroupofATRABDNFLY294002CDDPtheviabilityratedecreasedandtheapoptosisrateincreasedinSY5Ycellswhencompared砸tlltheATRABDNFCDDPgroup(P(005)；ButthesensibilityofSY5YcellstochemotherapydrugCDDPdecr

12、easedinthegroupofArRA斗BDNFU73122CDDPandATRABDNFU0126CDDPtheviabilityandtheapoptosisrateofthetwogroupshavenosignificantdifferencecomparedwiththeATRABDNFCDDPgroupf尸005)TheresultsofELISAimpliedthatMMP一9proteinlevelsinthelOn