花生四烯酸代謝促進(jìn)乳腺癌細(xì)胞增殖和遷移信號(hào)轉(zhuǎn)導(dǎo)途徑的研究.pdf

1、摘要摘要乳腺癌轉(zhuǎn)移是造成患者死亡的主要原因之一。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)，花生四烯酸(ArachidonicAcidAA)代謝可能與乳腺癌細(xì)胞高增殖和遷移的作用密切相關(guān)。本研究針對(duì)花生四烯酸代謝與上述高轉(zhuǎn)移乳腺癌細(xì)胞增殖和遷移信號(hào)通路的關(guān)系進(jìn)行了深入細(xì)致的研究。我們首先應(yīng)用蛋白質(zhì)組學(xué)方法對(duì)MCF7與LMMCF7細(xì)胞之間差異表達(dá)的蛋白進(jìn)行了檢測。經(jīng)二維電泳、質(zhì)譜分析檢測后，發(fā)現(xiàn)了8個(gè)有意義的蛋白質(zhì)點(diǎn)，其中甘油磷酸激酶1(phosphoglyc

2、eratekinase1)磷酸甘油醛異構(gòu)酶(triosephosphateisomerase)、烯醇酶(enolase)和磷酸甘油酸變位酶(phosphoglyceratemutase)等四個(gè)蛋白與細(xì)胞的糖代謝途徑有密切的關(guān)系。文獻(xiàn)報(bào)道花生四烯酸代謝是糖代謝的重要組成部分，上述結(jié)果從蛋白水平上提示從代謝可能與乳腺癌轉(zhuǎn)移相關(guān)。因此，我們進(jìn)一步研究了從代謝與高轉(zhuǎn)移乳腺癌細(xì)胞增殖和遷移信號(hào)途徑GioPLCPKCERK12MLCK加MLC的關(guān)系

3、。我們實(shí)驗(yàn)室發(fā)現(xiàn)，pERK12是上述乳腺癌細(xì)胞高增殖和遷移相關(guān)信號(hào)途徑的關(guān)鍵信號(hào)因子，為此本研究進(jìn)一步應(yīng)用免疫印跡驗(yàn)證了PERK12在LMMCF7MDAMB231及MCF7細(xì)胞中表達(dá)的差異。結(jié)果顯示，與低轉(zhuǎn)移的乳腺癌細(xì)胞系MCF7細(xì)胞相比，pERK12的水平在高轉(zhuǎn)移的乳腺癌細(xì)胞系LMMCF7和MDAMB231中明顯增高。為了進(jìn)一步研究AA代謝與LMMCF7細(xì)胞高轉(zhuǎn)移的關(guān)系，我們通過Westernblot實(shí)驗(yàn)，分別檢測了cPLA2COXL

4、OX和P450的抑制劑AACOCF3Indomethacin(Indo)NDGA和SKF525A對(duì)LMMCF7細(xì)胞中pERK12水平的影響。結(jié)果顯示，與AA釋放有關(guān)的cPLA2及COX和LOX抑制劑可明顯下調(diào)LMMCF7細(xì)胞中pERKI2的水平，表明AA代謝相關(guān)的cPLA2COX和LOX與上述乳腺癌高增殖和遷移信號(hào)途徑相關(guān)應(yīng)用RTPCR和免疫印跡實(shí)驗(yàn)對(duì)LMMCF7和MDAMB231與MCF7細(xì)胞進(jìn)行比較，結(jié)果發(fā)現(xiàn)高轉(zhuǎn)移細(xì)胞中cPLA2C

5、OX2以及5LOX的mRNA水平和蛋白水平的表達(dá)明顯上調(diào)，進(jìn)一步證實(shí)了上述結(jié)果。文獻(xiàn)報(bào)道Gi。和pERK12可以激活cPLA2，從而促進(jìn)AA的釋放和代謝。為了研究上述GioPLCPKCpERK12信號(hào)途徑是否與促進(jìn)AA的釋放和代謝有關(guān)，我們分別應(yīng)用上述各信號(hào)的抑制劑PTXCalphostinC和PD98059作用LMMCF7細(xì)AbstractAbstractMetastasisisoneofthereasonsofdeathofbrea

6、stcancerpatient.Ourlaboratorypreviouslyfoundthatarachidonicacidanditsmetabolites~closelyrelatedtotheabilityofhighproliferationandhighmetastasisofbreastcancercellswithighmetastasispotential.Inthisstudyweinvestigatedthesig

7、naltransductionpathwaysinvolvedinarachidonicacidanditsmetabolitesresultedinhighproliferationandhighmetastasisofbreastcancercells.FirstlyweusedproteomicsmethodstoinvestigatetheexpressionofdiferentproteinsbetweenLMMCF7andM

8、CF7cells.Twodimensionalelectrophoresesandmassspectrumresultsshowedthat8proteinsweresignificant.Ineightproteinsphosphoglyceratekinase1triosephosphateisomeraseenolaseandphosphoglyceratemutaseareallcloserelativetoglycolytic

9、signaltransductionpathways.Manypapersreportedthatarachidonicacidmetaboliteswereaveryimportantpartofglycolyticsignaltransductionpathways.Theseresultswerecuetousfurtherthatarachidonicacidmetaboliteswereprobablycloserelativ

10、e.tohighproliferationandmetastasissignaltransductionpathwayofLMMCF7cells.SowefurtherinvestigatedtherelationshipbetweenarachidonicacidmetabolitesandhighproliferationandmetastasissignaltransductionpathwayofLMMCF7cellsGioPL

11、CPKCERKI2MLCKpMLC.pERKI2isamainlycriticalsignalfactorinhighproliferationandmetastasissignaltransductionpathwayofLMMCF7cells.SoweinvestigatetheexpressionallevelofpERKI2inLMMCF7MCF7andMDAMB231cells.Westernblotresultsshowed

12、pERKI2expressionallevelisobviouslyhigherinLMMCF7andMCF7cellsthaninMCF7cells.InordertofurtherinvestigatetherelationshipofarachidonicacidmetabolitesandhighproliferationandmetastasissignaltransductionpathwayofLMMCF7cellswee