Ezrin siRNA對骨肉瘤細胞株SaOS-2 ezrin表達及生物學(xué)行為的影響.pdf

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

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

3、殖能力，以MillicellPCF侵襲小室研究細胞體外侵襲、運動性變化，并檢測細胞異質(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干擾對細胞增殖和侵襲能力無影響，但能抑制細胞粘附和運動能力。結(jié)論特異性siRNA可以抑制ezrin基因的表達

4、，抑制細胞粘附和運動，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；實時定量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