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1、RNA干擾MBP-1基因?qū)ξ赴┘?xì)胞SGC-7901增殖影響耿晢1, 姚海燕1,韓躍武1,(1蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院生物化學(xué)及分子生物學(xué)研究所甘肅蘭州730000)基金項(xiàng)目：蘭州大學(xué)“中央高校基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金”自由探索項(xiàng)目，項(xiàng)目編號(hào)lzujbky-2012-187.【摘要】目的：探討MBP-1基因表達(dá)沉默對(duì)胃癌細(xì)胞株SGC-7901細(xì)胞增殖影響。方法：實(shí)驗(yàn)分3組：空白對(duì)照組（未轉(zhuǎn)染胃癌細(xì)胞）、陰性對(duì)照組（轉(zhuǎn)染錯(cuò)義序列組）和干擾組（轉(zhuǎn)染MBP-1shRNA組）.設(shè)計(jì)2條針對(duì)MBP-1基因的小干擾RNA片段及1條
2、陰性對(duì)照siRNA,并構(gòu)建入pSIREN-retroQ質(zhì)粒。將構(gòu)建的重組pSIREN-retroQ質(zhì)粒通過Lipofectamine2000脂質(zhì)體轉(zhuǎn)染胃癌SGC-7901細(xì)胞，通過嘌呤霉素篩選構(gòu)建穩(wěn)轉(zhuǎn)株細(xì)胞。RealtimeRT-PCR和westernblot分別檢測(cè)MBP-1表達(dá)。MTT法對(duì)MBP-1干擾后對(duì)SGC-7901細(xì)胞增殖的影響進(jìn)行檢測(cè)。結(jié)果：通過PCR擴(kuò)增陽性克隆及測(cè)序，說明已成功構(gòu)建MBP-1干擾及對(duì)照重組pSIREN-retroQ質(zhì)粒。通過Lipofectamine2000脂質(zhì)體將重組質(zhì)粒轉(zhuǎn)
3、染胃癌SGC-7901細(xì)胞，并通過嘌呤霉素篩選兩周，說明已成功構(gòu)建MBP-1干擾及對(duì)照SGC-7901穩(wěn)轉(zhuǎn)株細(xì)胞。通過RealtimeRT-PCR檢測(cè)，干擾組MBP-1mRNA相對(duì)表達(dá)量與空白對(duì)照組相比都顯著下調(diào)（P＜0.05）。通過westernblot檢測(cè)檢測(cè)MBP-1蛋白表達(dá)，干擾組MBP-1的表達(dá)量與空白對(duì)照組相比也都顯著下調(diào)（P＜0.05）。MTT法檢測(cè)結(jié)果表明，MBP-1干擾組細(xì)胞在24、48、72、96和120小時(shí)增殖能力比空白對(duì)照組都有顯著的升高（P＜0.05）。結(jié)論：MBP-1基因表達(dá)能抑制
4、胃癌細(xì)胞SGC-7901的增殖，推測(cè)MBP-1基因可能成為胃癌基因治療的一個(gè)新靶點(diǎn)。【關(guān)鍵詞】胃癌細(xì)胞；RNA干擾；MBP-1；細(xì)胞增殖；EffectsofMBP-1geneexpressiondownregulationbyRNAinterferenceontheproliferationofgastriccancercelllineSGC-7901.GengZhe1,YaoHaiyan1,HanYuewu1,（1Instituteofbiochemistryandmolecularbiology,Lanz
5、houmedicalcollege,LanzhouUniversity,Lanzhou730000,China）【Abstract】Objective:Toinvestigatetheeffectsofc-mycpromoterbindingprotein(MBP-1)geneontheproliferationinvitroinhumangastriccancercelllineSGC-7901.Methods:Cellsdividedintothreegroups：blankcontrolgroup(un
6、transfectedcells)，negativegroup(cellstransfectedwithmissensesequence)andexperimentalgroup(cellstransfectedwithMBP-1shRNA)．TwoMBP-1shRNAsequencesandonenegativecontrolshRNAsequenceweredesigned,synthesizedandclonedintopSIREN-retroQplasma.Thentherecombinantplas
7、midswereconstructedandtransfectedintohumangastriccancerSGC-7901cellsbyLipofectamine2000．Afterselectionofcellswithpuromycinfortwoweeks,stablecelllineSGC-7901expressinggeneofMBP-1specificshRNAwasobtained.TheexpressionsofMBP-1mRNAandproteininSGC-7901weredeterm
8、inedbyRealtimePCRandWesternblotting,respectively.TheeffectsofalteredexpressionofMBP-1oncellproliferationweremeasuredbyMTTcellproliferationassay.Results:PCRandsequencingindicatedthattherecombinantplasmi
