沉默乙酰肝素酶對(duì)胃癌侵襲轉(zhuǎn)移影響的體外研究

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1、中文摘要目的采用RNA干擾技術(shù)探討沉默乙酰肝素酶(heparanaseHPa)對(duì)人胃癌細(xì)胞株SGC一7901轉(zhuǎn)移和移動(dòng)能力的影響。方法根據(jù)人HPa基因序列設(shè)計(jì)4個(gè)小干擾RNA(smallinterferingRNA，sirtNA)靶位點(diǎn)(HPal521、HPal324、HPal085、HPallll)，并構(gòu)建了4個(gè)針對(duì)靶位點(diǎn)的一個(gè)載體編碼一個(gè)短發(fā)夾狀RNA(shRNA)質(zhì)粒表達(dá)載體，同時(shí)合成了1個(gè)陰性對(duì)照質(zhì)粒表達(dá)載體(shNC)和1個(gè)陽性對(duì)照質(zhì)粒表達(dá)載體(shhGAPDH)，采用脂質(zhì)體介導(dǎo)轉(zhuǎn)染人胃癌細(xì)胞株SGC一7901，用Western—blot和RT—PCR檢測(cè)轉(zhuǎn)染前后乙酰肝素酶

2、蛋白和mRNA表達(dá)的變化，選出對(duì)乙酰肝素酶抑制效果最顯著的質(zhì)粒作為研究對(duì)象，利用其轉(zhuǎn)染細(xì)胞SGC一7901，并用G418篩選穩(wěn)定轉(zhuǎn)染的細(xì)胞，擴(kuò)增培養(yǎng)，應(yīng)用Transwell小室侵襲試驗(yàn)和劃痕損傷實(shí)驗(yàn)分別研究轉(zhuǎn)染前后細(xì)胞SGC一7901侵襲、轉(zhuǎn)移能力的變化。結(jié)果脂質(zhì)體介導(dǎo)質(zhì)粒HPal521、HPal324、HPal085、HPallll轉(zhuǎn)染細(xì)胞后，轉(zhuǎn)染后細(xì)胞乙酰肝素酶mRNA的表達(dá)量(HPamRNA光密度值與相應(yīng)內(nèi)參之比)分別為0．83s0．03、0．78±0．05、0．36s0．01、0．67±0．04，乙酰肝素酶蛋白表達(dá)量(HPa蛋白表達(dá)光密度值與相應(yīng)內(nèi)參之比)分別為0．87±0．

3、01、0．76±0．03、0．38±0．01、0．69s0．03D質(zhì)粒HPal085對(duì)乙酰肝素酶的抑制作用較其它質(zhì)粒顯著，質(zhì)粒HPal085轉(zhuǎn)染SGC7901細(xì)胞后并用C,418篩選后，在Transwell小室侵襲試驗(yàn)中，轉(zhuǎn)染組與正常對(duì)照組侵襲的細(xì)胞數(shù)分別為44±15．22和289．9±18．11，轉(zhuǎn)染組侵襲細(xì)胞數(shù)較正常對(duì)照組顯著減少(P

4、詞RNA小分子干擾乙酰肝素酶SC,C一7901TheEffectofHeparanaseGeneSilenceontheInvasionandMetastasisofhumangastriccarcinomacellinVitroAbstractObjectiveToevaluatetheeffctofsilenceheparanaseontheinvasivenessinhumangas—triccarcinomacelllineSGC一7901usingRNAinterferenceteehnolog．MethodsFourtarge—tedlocations(HPal521、H

5、Pal324、HPal085、andHPalI11)weredesignedandfourplasmidvectorsexpressingshorthairpinRNA(shRNA)tartgetingHPawereconstructedaccordingtotranscriptionRNAlocationofhumanHPagenesequenceanddesigningprincipleofsmallin—terferenceRNA(siRNA)．Alsoanegativeandapositivecontrolplasmidvectorswerecon-stmcted．Theyw

6、eretransfectedintohumangastriccarcinomaSGC一7901ceilsmediatedbycationicliposomelipofectin：HPamRNAandproteinexpressionlevelsweredetectedbyRT—PCRandWesternblotting，respectively．TheplasmidthatremarkablyinhibitedheparanaseWasdecided．Aftertransfectedwiththisplasmid，positivecellcloneswereselectedwithG

7、418andamplefied．Stabletransfectedceilswereobtainedandroutinelymaintainedinselectionmediumtoavoidovergrowthofnon—transfectedcells．Invasiveandmobilitypowerwereevaluatedbe—foreandaftertransfactionbythemembranceinvasionculturesystem(T