資源描述:
《靶向fak的sirna對(duì)喉癌細(xì)胞增殖及侵襲能力的抑制作用》由會(huì)員上傳分享,免費(fèi)在線閱讀,更多相關(guān)內(nèi)容在工程資料-天天文庫(kù)。
1、靶向FAK的siRNA對(duì)喉癌細(xì)胞增殖及侵襲能力的抑制作用張潛英,葉琳,余曉燕,沈娜,鄧碧,吳曉松,陳鴻雁(重慶醫(yī)科大學(xué)附屬第一醫(yī)院耳鼻咽喉科,重慶400016)摘要:目的運(yùn)用RNA干擾(RNAinterference,RNAi)技術(shù)阻斷喉癌Hep-2細(xì)胞株中FAK基因的表達(dá),并研究FAK基因沉默后對(duì)Hep-2細(xì)胞株產(chǎn)生的影響。方法針對(duì)FAK基因,構(gòu)建2條干擾重組質(zhì)粒FAK-pGenesil-CH1和FAK-pGenesil-CH2,1條陰性對(duì)照重組質(zhì)粒FAK-pGenesil-HK;在脂質(zhì)體的介導(dǎo)下轉(zhuǎn)染喉癌He
2、p-2細(xì)胞株,用RT-PCR和Westernblot分析FAKmRNA及蛋白的表達(dá);MTT法、流式細(xì)胞技術(shù)及體外侵襲實(shí)驗(yàn)測(cè)定干擾重組質(zhì)粒對(duì)Hep-2細(xì)胞株的增殖、侵襲能力的影響。結(jié)果重組干擾質(zhì)粒能有效地阻斷Hep-2細(xì)胞株中FAK基因在mRNA和蛋白水平上的表達(dá)(P<0.05);重組質(zhì)粒轉(zhuǎn)染Hep-2細(xì)胞后,細(xì)胞增殖抑制率分別為58.34%、52.78%,與對(duì)照組比較差別有統(tǒng)計(jì)學(xué)意義(P<0.05);干擾組G0~G1期細(xì)胞增多,S期細(xì)胞數(shù)量明顯減少(P<0.05);FAK-pGenesil-CH1、FAK-pG
3、enesil-CH2作用Hep-2細(xì)胞株48h后,Hep-2細(xì)胞穿過(guò)Matrigel膜的個(gè)數(shù)分別為(56.0±6.7)、(51.0±5.1),與陰性對(duì)照組及正常對(duì)照組[分別為(152.0±9.4)和(139.0±8.1)]比較有統(tǒng)計(jì)學(xué)差別(P<0.05)。結(jié)論干擾重組質(zhì)粒能有效地抑制轉(zhuǎn)染Hep-2細(xì)胞株的FAKmRNA及蛋白的表達(dá),并能抑制Hep-2細(xì)胞株的增殖及侵襲能力。關(guān)鍵詞:FAK;siRNA;RNA干擾;Hep-2細(xì)胞株;增殖;侵襲中圖法分類號(hào):R739.65文獻(xiàn)標(biāo)識(shí)碼:AEffectofFAKgene
4、silencingonproliferationandinvasioninofHep-2cellsZHANGQian-ying,YELin,YUXiao-yan,SHENNa,DENGBi,WUXiao-song,CHENHong-yan(DepartmentofOtolaryngology,theTheFirstAffilatedHospital,,ChongqingMedicalUniversity,,Chongqing400016,,China)Abstract:objectiveObjectiveToo
5、bservetheeffectsofRNAi-mediatedFAKgeneonsilencinginonlaryngealcarcinomaHep-2cells..MethodsConstructrRecombinantplasmidsgeneratingshorthairpinRNAwereconstructed,includingtwointerferentialsequencesFAK-pGenesil-CH1、,FAK-pGenesil-CH2andonenegativecontrolsequence
6、FAK-pGenesil-HK.Hep-2cellsweretransfectedwithrecombinantplasmids..FAKmRNAandFAKproteinwereanalyzedrespectivelybyRT-PCRandWesternblotting,respectively..Cellproliferation、,cellcycleandinvasionofHep-2cellswereobservedbyMTTassay、,flowcytometry(FCM)andtranswellin
7、vasionassay,respectively.ResultsRecombinantplasmidsofFAK-pGenesil-CH1andFAK-pGenesil-CH2downregulatedtheexpressionsofFAKmRNAandprotein(P<0.05);.TheproliferationinhibitionratioinHep-2cellsreachedto58.34%、and52.78%,%48hourshaftertransfectionofrecombinantplasmi
8、dsofFAKpGenesil-CH1、andFAK-pGenesil-CH2,whilethatthecontrolwas8.34%inthecontrol(P<0.05)(P<0.05).;FCManalysisshowedthatmMorecellsstayarrestedatinG0-/G1phaseinthetransfectedgroupsthancontrols;.The