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1、萬方數(shù)據(jù)·20·.口腔頜面腫瘤防治.蛋白多糖與涎腺腺樣囊性癌增殖的關(guān)系石宏王潔董福生王旭李荷香【摘要】目的探討蛋白多糖(proteoglycans)合成的阻抑對人涎腺腺樣囊性癌ACC.M細(xì)胞增殖的影響。方法構(gòu)建靶向人木糖基轉(zhuǎn)移酶-I(xylosyltransferase—I,XT—I)基因的短發(fā)卡狀RNA(shorthairpinRNA,shRNA)真核表達載體shRNA—WJ3,轉(zhuǎn)染人涎腺腺樣囊性癌高轉(zhuǎn)移株ACC.M細(xì)胞,通過G418篩選穩(wěn)定轉(zhuǎn)染的細(xì)胞,采用實時定量聚合酶鏈反應(yīng)和蛋白質(zhì)印跡法測定shRNA.WJ3的基因沉默效果;檢測各組細(xì)胞蛋白多糖合成分泌水平的變化
2、。采用甲基嚷唑基四唑法(M1Tr)檢測細(xì)胞增殖情況;流式細(xì)胞術(shù)測定細(xì)胞周期改變。結(jié)果shRNA-WJ3轉(zhuǎn)染ACC.,M細(xì)胞后,細(xì)胞中XT.I基因的mRNA和蛋白表達抑制率分別為83.70%和79.60%;穩(wěn)定轉(zhuǎn)染shRNA-WJ3的ACC.M.WJ3細(xì)胞蛋白多糖分泌顯著降低(P<0.01),抑制率為49.7l%一54.59%。MTr法檢測結(jié)果表明:蛋白多糖合成阻抑后,ACC-M細(xì)胞增殖活性明顯降低;ACC—M-WJ3細(xì)胞與對照組細(xì)胞相比:s期細(xì)胞數(shù)日減少;G。一Go期細(xì)胞數(shù)目增加,差異有統(tǒng)計學(xué)意義(P<0.01)。結(jié)論蛋白多糖合成分泌的阻抑能有效抑制ACC—M細(xì)胞的增
3、殖?!娟P(guān)鍵詞】癌,腺樣囊性;涎腺;蛋白多糖RelationshipbetweenproteoglycansandproliferationofhumansalivaryadencidcysticcarcinomasH
4、Hong。WAl、Gj沈’,DONGFu—sheng,WANGXu.uHe—xiang.‘DepartmentofOralPathology。CollegeofStomatology,脅beiMedicalUniversity.Sh“iazhuang050017,ChinaCorrespondingauthor:WANGJie,Email:wangiie
5、phd@163.COIn,Td:0086—3l1—86265780【Abstract】ObjectiveToinvestigatetheeffectofdown·regulatedproteoglycansontheproliferationofhumansalivaryadenoidcysticcarcinoma(SACC).MethodsTheshorthairpinRNA(shRNA)plasmidsilencinghumanxylosyhransferase—I(XT-I)geneWflftconstructedandnamedshRNA.WJ3.Adenoi
6、dcysticcarcinomacellswithhighmetastatictendency(ACC.M)weretransfectedbyshRNA.WJ3.TheplasmidshRNA—HKnottargetinganyhumangenewastransfectedintoACC.Mcellsused鼬negativecontroLAfter48hoftransfection.t}IepositivecellswerescreenedbyC-418toisolatethestabletransfectedceils.Real-timePCRandWestern
7、blottingwereusedtotestthegenesilence。andtheproteoglycanscontentsofthecellsweredetected.ThestablecelllinesilencedXT-IWasnamedACC-M—WJ3.M33'assaywa$performedtodetectthecellproliferation.ThecellcycleWaSanalyzedbyflowcytometry.ResultsShRNA.WJ3showedpowerfulRNAinterferenceandgenesilenceofXT-
8、I.Theinhibitionratewas83.70%ofmRNAexpressionand79.60%ofproteinexpressionrespectively.111econtentofproteoglycansinACC-M—WJ3Wasdown-regulatedby49.71%-54.59%.TheresultsofMTrassayshowedthattheeellgrowthWasinhibitedsignificantly.SphrasedecreasedandGI—GophraseincreasedingroupACC—M-WJ