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1、腫瘤2009年4月第29卷第4期TumorVol.29,No.4,April,2009·345·DOI:10.3781/j.issn.100027431.2009.04.011BasicResearch·基礎(chǔ)研究livin基因沉默對(duì)人惡性黑素瘤A375細(xì)胞周期和凋亡的影響王琳琳,鄭洪,唐薇薇,厲國慧,安文波(遵義醫(yī)學(xué)院附屬醫(yī)院病理科,遵義563003)[摘要]目的:探討小干擾RNA(smallinterfeningRNA,siRNA)沉默livin基因?qū)θ藧盒院谒亓鯝375細(xì)胞周期和凋亡的影響。方法:構(gòu)建2個(gè)針對(duì)人源livin基因的siRNA表達(dá)質(zhì)粒,并通過脂質(zhì)體介導(dǎo)
2、轉(zhuǎn)染人惡性黑素瘤A375細(xì)胞株,利用熒光定量PCR檢測(cè)livinmRNA的表達(dá)情況,AnnexinⅤ2PEFCM法檢測(cè)A375細(xì)胞凋亡情況,PI單染FCM法檢測(cè)細(xì)胞周期情況。結(jié)果:成功構(gòu)建livinsiRNA表達(dá)質(zhì)粒,轉(zhuǎn)染siRNA21和siRNA22后48h的A375細(xì)胞中l(wèi)ivinmRNA表達(dá)量較空白對(duì)照組分別下降17%和42%,72h分別下降71%和61%。轉(zhuǎn)染siRNA21和siRNA22后48h的A375細(xì)胞凋亡率(29.45%和33.25%)及72h細(xì)胞凋亡率(33.52%和38.18%)均高于空白組(7.95%和9.70%)(P<0.05)。siRNA2
3、2可引起G0/G1期升高,S期下降(P<0.05),siRNA21作用不明顯(P>0.05)。結(jié)論:siRNA21、2體外均能有效抑制livinmRNA的轉(zhuǎn)錄,促進(jìn)人惡性黑素瘤A375細(xì)胞凋亡。siRNA22表達(dá)質(zhì)粒能使人惡性黑素瘤A375細(xì)胞周期出現(xiàn)G0/G1期阻滯,細(xì)胞增殖受到抑制。[關(guān)鍵詞]黑色素瘤,實(shí)驗(yàn)性;RNA干擾;細(xì)胞凋亡;細(xì)胞周期;基因,livin[中圖分類號(hào)]R739.5[文獻(xiàn)標(biāo)志碼]A[文章編號(hào)]100027431(2009)0420345205EffectsoflivingenesilencingbysiRNAonapoptosisandcellc
4、ycleofhumanmalignantmelanomaA375cellsWANGLin2lin,ZHENGHong,TANGWei2wei,LIGuo2hui,ANWen2bo(DepartmentofPathology,AffiliatedHospitalofZunyiMedicalCollege,Zunyi563003,China)[ABSTRACT]Objective:ToexploretheeffectsoflivingenesilencingbysmallinterfeningRNA(siRNA)ontheapoptosisandcellcycleofhu
5、manmalignantmelanomaA375cells.Methods:TwosiRNAeukaryoticexpressionvectortargetingthelivingenewerecon2structedbygenerecombination,andthentransfectedintohumanmalignantmelanomaA375cellsmediatedbyliposome.ExpressionleveloflivinmRNAwasassayedbyreal2timefluorescencequantitativePCRtechnology.T
6、heapoptosiswasdetectedbyAnnexinV2PEflowcytometry(FCM)andthecellcyclewasdeterminedbyFCMwithsinglePIstaining.Results:Theexpressionvectorstargetinglivinweresuccessfullyconstructed.ExpressionoflivinmRNAinA375cellswasdecreasedby17%and42%after482htransfectionofsiRNA21andsiRNA22comparedwiththe
7、blankcontrolgroup;anddecreasedby71%and61%after722htransfection.TheapoptoticratiosofA375cellswere29.45%and33.25%after482htransfectionofsiRNA21andsiRNA22,respectively.Theywere33.52%and33.18%after722htransfectionofsiRNA21andsiRNA22,respectively.Allwerehigherthanthoseinblankcontrol
