Ezrin siRNA對(duì)骨肉瘤細(xì)胞株SaOS-2 ezrin表達(dá)及生物學(xué)行為的影響.pdf

1、第二軍醫(yī)大學(xué)博士學(xué)位論文EzrinsiRNA對(duì)骨肉瘤細(xì)胞株SaOS2ezrin表達(dá)及生物學(xué)行為的影響姓名：楊洪平申請(qǐng)學(xué)位級(jí)別：博士專業(yè)：外科學(xué)（骨外）指導(dǎo)教師：張少成20050501EzrinsiRNA對(duì)骨肉瘤細(xì)胞株SaOS2ezrin表達(dá)及生物學(xué)行為的影響【摘要】目的應(yīng)用特異性siRNA(小干涉RNA)抑制人骨肉瘤細(xì)胞ezrin基因表達(dá)，觀察其對(duì)SAOS2細(xì)胞生物學(xué)特性的影響。方法設(shè)計(jì)并化學(xué)合成2條針對(duì)ezrin蘸因的2IntsiRN

2、A，以150nM轉(zhuǎn)染SaOS2細(xì)胞，應(yīng)用實(shí)時(shí)定量PCR檢測(cè)ezrinmRNA表達(dá)，篩選出抑制效率最高的siRNA：再分別以／fi涮濃度的siRNA轉(zhuǎn)染人骨肉瘤細(xì)胞，應(yīng)用實(shí)時(shí)定量PCR檢測(cè)ezrinmRNA表達(dá)：再以這條siRNA及其最佳抑制濃度轉(zhuǎn)染細(xì)胞，抽提孵育24、48、72、96h細(xì)胞總RNA及蛋白質(zhì)，采用實(shí)時(shí)熒光定量PCR和Westernblot檢測(cè)ezrinmRNA及蛋白質(zhì)表達(dá)情況。應(yīng)用MTT法檢測(cè)siRNA干擾后骨肉瘤細(xì)胞的增

3、殖能力，以MillicellPCF侵襲小室研究細(xì)胞體外侵襲、運(yùn)動(dòng)性變化，并檢測(cè)細(xì)胞異質(zhì)粘附的改變情況。結(jié)果siRNA抑制效率在9375～150nM范圍內(nèi)呈濃度依賴性增高，以siRNA—l150riM最顯著；轉(zhuǎn)染siRNA1的SaOS2細(xì)胞24、48、72、96hezrinmRNA和蛋白質(zhì)下調(diào)，分別以48h、72h最顯著。siRNA干擾對(duì)細(xì)胞增殖和侵襲能力無影響，但能抑制細(xì)胞粘附和運(yùn)動(dòng)能力。結(jié)論特異性siRNA可以抑制ezrin基因的表達(dá)

4、，抑制細(xì)胞粘附和運(yùn)動(dòng)，從而抑制骨肉瘤轉(zhuǎn)移?！娟P(guān)鍵詞】ezrin；小干擾RNA；RNA干擾；骨肉瘤；侵襲；轉(zhuǎn)移；實(shí)時(shí)定量PCREffectofspecificsiRNAtargetingagainstezringeneonezrinexpressionandthebiologicalbehaviorofSaOS2OSteosareomacellline【Abstract】objective：Toinvestigatetheefl’ecto

5、fspecificsmallinterferingRNA(siRNAltargetingezringeneonthebiologicaltraitsofSaOS2ceilsMethods：TwosiRNAstargetingezringeneweredesigned，chemicallysynthesized，andtransfectedintotheSAOS一2cellsTheexpressionofezrinmRNAinSAOS一2

6、cellswasevaluatedbyreal—timeRTPCRTheoptimalsiRNAWastransfectedintoSaOS一2cellsatdifierentconcentration，mRNAlevelsweredetectedusingrealtimeRTPCRThesiRNAwithconsequentconcentrationwhichhadthemaximuminhibitionratewasselected

7、andtransfectedintoSaOS一2cellsTotalRNAandproteinofthecellsafter24h，48h，72h，and96hincubationwereextracted，respectivelyTheexpressionezrininmRNAandproteinlevelwereassessedbyrealtimeRTPCRandWesternblot，respectivelyThecellular

8、proliferationwasmeasuredbytheMTTassayTheMotilityandinvasivepotentialofcellsWasevaluatedinMillicellPCFinsertwithuncoatedorMatrigelcoatedfiltersof8unlporesCellularadhesiontomatricesofpurifiedcollagenIandMatrigelwasquantifi

9、edResults：DegreeofdownregulationinezrinmRNAincreasedinadosedependentmannerwithin9，375150mnol／LThemaximuminhibitoryrateWasfoundin150nmol／L，TheexpressionofezrinatmRNAlevelandproteinlevelWaSreducedTheoptimalsilencingeffecta

10、tmRIgAandproteinlevelwasat48hand72hposttransfection，respectivelyNodifferenceincellproliferationandinvasionabilitywasfoundbetweentheexperimentalgroupandcontrolgroupComparedwiththecontrolgroup，thecelladhesionandmotilitypot

11、entialweresignificantlydecreasedinexperimentalgroupConclusion：ThespecificsiRNAcouldknockdowntheexpressionofezrinandinhibitscelladhesionandmotilityItmayplayallinhibitoryroleinthecellularattachmentandmotilitypotentialinthe