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1、中華肝膽外科雜志2014年8月第2O卷第8期ChinJHepatobiliarySurg，August2014，Vo1．20，No．8·599··實(shí)驗(yàn)研究·EphA7基因沉默對人肝癌細(xì)胞SMMC一7721裸鼠移植瘤生長的影響張弓李捷翟文龍郭文治趙永福張水軍【摘要】目的探討RNA干擾抑制EphA7基因?qū)θ烁伟┘?xì)胞SMMC一7721裸鼠皮下移植瘤生長的影響。方法EphA7小干擾RNA(siRNA)重組質(zhì)粒采用脂質(zhì)體介導(dǎo)法轉(zhuǎn)染肝癌細(xì)胞SMMC一7721，同時設(shè)空載體轉(zhuǎn)染組、未轉(zhuǎn)染組和對照組。對照組不注射細(xì)胞，僅注入磷酸鹽緩沖液(PBS)做對照。裸鼠移植瘤模型采用左上肢皮下注射的方法建立。應(yīng)用實(shí)時P
2、CR、免疫組化和蛋白印跡方法檢測移植瘤組織中EphA7基因和蛋白的表達(dá)情況，同時比較成瘤時間、腫瘤體積和重量等。結(jié)果注射細(xì)胞后9～12天，除對照組外其余各組均可在注射部位發(fā)現(xiàn)肝癌移植瘤形成。移植瘤形成后第35天，與未轉(zhuǎn)染組和空載體轉(zhuǎn)染組比較，轉(zhuǎn)染組移植瘤生長速率、腫瘤體積和重量均明顯降低(P<0．05)。轉(zhuǎn)染干擾載體pRNA—siEphA7對裸鼠肝癌移植瘤的的抑瘤率為55％。免疫組化結(jié)果顯示，與未轉(zhuǎn)染組和空載體轉(zhuǎn)染組比較，轉(zhuǎn)染組移植瘤組織中EphA7蛋白陽性染色的細(xì)胞較少，染色較淺，呈淺黃色。實(shí)時PCR和蛋白印跡結(jié)果顯示，與未轉(zhuǎn)染組和空載體轉(zhuǎn)染組比較，轉(zhuǎn)染組移植瘤組織中EphA7基因和蛋白的
3、表達(dá)明顯低于未轉(zhuǎn)染組和空載體轉(zhuǎn)染組，差異有統(tǒng)計學(xué)意義(P<0．05)。結(jié)論利用siRNA抑制EphA7的表達(dá)可減緩肝癌細(xì)胞SMMC-7721在活體動物體內(nèi)的生長，有望成為肝癌基因治療的靶點(diǎn)?！娟P(guān)鍵詞】肝細(xì)胞癌；裸鼠移植瘤；RNA干擾；基因EphA7；基因療法RNAinterferencetargetingEphA7inhibitsgrowthofSMMC-7721cellxenograftinnudemiceZhangGD，LiJie，ZhaiWenlong，GuoWenzhi，Zhaon，ZhangShuijun．DepartmengofHepatobiliary-pan—creaticS
4、urgery，theFimtAffiliatedHospitalofZhengzhouUniversity，Zhengzhou450052，ChinaCorrespondingauthor：ZhangShuijun，Email：zhangshuijun@M．edu．cn【Abstract】0bjeetiveToinvestigatetheeffectsofRNAinterferencetargetingEphA7geneonthegrowthofSMMC-7721cellxenograftinnudemice．MethodsRecombinantplasmidofEphA7gene—targ
5、e—tingsiRNAwastransfectedintohepaticcancerSMMC-7721cellsbyLipofectamine2000．comparingwiththeemptyvectortransfectedgroup，untransfeetedgroupandcontrolgroup．ThenudemicetumormodelwasestablishedbysubcutaneousiniectionofhepaticcancerceUsintheleftupperlimbofthemice．ControlgroupwasiniectedwithPBSasblank．Re
6、al—timePCR。immunohistocbemistryandWesternblotwereemployedtodetectthemRNAandproteinexpressionsofEphA7intumortissues．Thetumorformationtime，tumormassandweightoftumorwerealsoconsideredintheanalysis．ResultsAbout9—12daysaftertheinjectionoftumorcells，thexenografttumorformationcanbeobservedaroundtheinjecti
7、onsiteexceptthecontrolgroup．35daysaftertumorformation，therewereobviousdecreasesinthetumorgrowthrate，tumormass，aswellastumorweightintransfeetedgroup，comparingwithemptyvectortransfectedgroupanduntransfectedgr
