靶向sirna阻斷nf-kb信號通路對神經(jīng)母細(xì)胞瘤侵襲轉(zhuǎn)移的影響

《靶向sirna阻斷nf-kb信號通路對神經(jīng)母細(xì)胞瘤侵襲轉(zhuǎn)移的影響》由會員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。

1、靶向siRNA阻斷NF—KB信號通路對神經(jīng)母細(xì)胞瘤侵襲轉(zhuǎn)移的影響中文摘要目的探討重組慢病毒介導(dǎo)siRNA(小干擾RNA)靶向阻斷NF—KB信號通路對人神經(jīng)母細(xì)胞瘤QDD刪，細(xì)胞侵襲轉(zhuǎn)移的影響。方法根據(jù)人p65基因序列，設(shè)計針對基因不同靶點的siRNAl、siRNA2、siRNA3、siRNA44條siRNA序列及其對照序列NC，慢病毒介導(dǎo)轉(zhuǎn)染QDDQ—NM。，采用實時熒光定量PCR(RT—PCR)和Westernblot法從設(shè)計的4條干擾序列中篩選出siRNAI序列在轉(zhuǎn)染72h干擾效果最好。在轉(zhuǎn)染72h后，采用RT-PCR和Westernblot法檢測NF—KB下游基

2、因CXCR4、VEGFmRNA轉(zhuǎn)錄和蛋白表達(dá)情況；Transwell小室檢測各組間瘤細(xì)胞的轉(zhuǎn)移侵襲能力。結(jié)果轉(zhuǎn)染72h后，PT—PCR顯示，siRNAI—NF—KBCXCR4、VEGFmRNA表達(dá)分別為空白對照組0．66倍、0．76倍，差異有統(tǒng)計學(xué)意義(P<0．05)；Westernblot顯示，siRNA卜NF—KBCXCR4、vEGF蛋白表達(dá)分別為空白對照組0．67倍、0．76倍，差異有統(tǒng)計學(xué)意義(P<0．05)；Transwell小室顯示，I(實驗組)、II(空載對照組)、HI(空白對照組)穿過Matrigel膠的細(xì)胞數(shù)分別為34．6000±3．9749；45．

3、6000±2。3021；54．8000±2．3874，各組問差異有統(tǒng)計學(xué)意義(P

4、ToexploretheeffectsoftheNF-kappaBpathwayblockedbysmallinterferingRNA(siRNA)mediatedbYlentivirusontheinvasionandmetastasisofhumanneuroblastomacancercellQDDQ—NMl．MethodsAccordingtohumanp65genesequences，4siRNAstargetingdifferenttargetsofp65andNCcontrolsequencesweresynthesizedandtmusfectedi

5、ntoQDDQ刪1．Andthebestsequenceoftransfectioninterferenceeffectwasscreenedbyreal-timefluorescentquantitativereversetranscription-polymerasechainreaction(RT-PCR)andWesternblot．TheinterferingeffectofsiRNA1wasoptimalamong4siRNAsafter72h．Atthistime，theexpressingofNF-r33downstreamgenesCXCR4andV

6、EGFatmRNAandproteinlevelswerealsodetected．ThecelluarinvasionandmetastasiscapabilityofdifferentgroupswereevaluatedbyTranswellassay．ResultsAt72hpost-transfection,comparedwithcontrolgroups，RT-PCRshowedthattheexpressionsofCXCR4andVEGFmRNAtargetingsiRNA1respectivelydecreased(0．66times，0．76ti

7、mesP<0．05)．AndWesternblotindicatedthattheexpressionofCXCR4andVEGFproteinrespectivelydecreased(O．67times，0．76times，P<0．05)；TranswelldemonstratedthatthenumberofcellspassingthroughMatrigelrubberofsiRNA1interference，transfectioncontrolandcontrolgroupswere34．6000士3．9749：45．6000士2．30