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《sirna沉默hif-1α對缺氧培養(yǎng)的人脈絡(luò)膜黑色素瘤細(xì)胞epo表達(dá)的影響》由會員上傳分享,免費(fèi)在線閱讀,更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、siRNA沉默?F-1Q對缺氧培養(yǎng)的人脈絡(luò)膜黑色素瘤細(xì)胞EPO表達(dá)的影響摘要目的:采用陽離子脂質(zhì)體介導(dǎo)的轉(zhuǎn)染方法,以RNA干擾為手段,研究HIF—lU—siRNA對缺氧培養(yǎng)的人眼脈絡(luò)膜黑色素瘤細(xì)胞(OCM一1)轉(zhuǎn)染前后缺氧條件下HIF—l0和EPO的表達(dá),初步探討HIF一1a在缺氧條件下對人眼脈絡(luò)膜黑色素瘤的調(diào)控作用。方法:『F常組OCM-l細(xì)胞采用含10%胎牛血清、1%雙抗(青一鏈霉素混合液)的DMEM高糖培養(yǎng)基培養(yǎng),將細(xì)胞置37℃、5%C02細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng),取處于對數(shù)期生長的細(xì)胞用于實(shí)驗(yàn)研究。缺氧組OCM一1細(xì)胞在
2、原培養(yǎng)基中加入CoCl2至濃度為100iamol/L用于模擬腫瘤內(nèi)部乏氧微環(huán)境。并將缺氧組細(xì)胞分為單純?nèi)毖踅M、干擾組、陰性對照組、陽性對照組、脂質(zhì)體對照組;設(shè)計(jì)并合成siRNA(siRNA+陰性對照+陽性對照),并在基因庫進(jìn)行Blast同源性分析。利用陽離子脂質(zhì)體Lipofectamine⑩2000Reagent為載體將合成的siRNA轉(zhuǎn)入脈絡(luò)膜黑色素瘤細(xì)胞OCM—l內(nèi)。RT—PCR檢測OCM—l細(xì)胞HIF一1U及EPO在RNA水平上的表達(dá)情況,Westernblot檢測HIF一1U及EPO蛋白表達(dá)情況。結(jié)果:缺氧組各組
3、之問相比,陽性對照組B—actinmRNA表達(dá)下降(P0.05),蛋白表達(dá)顯著升高(P0.05)。結(jié)論:】.在缺氧狀態(tài)下OCM1細(xì)胞HIF一1alnRNA表達(dá)無明顯變化,蛋白水平表達(dá)升高;EPO的mRNA和蛋白表達(dá)量均增加。2.合
4、成的HIF一1a—siRNA通過轉(zhuǎn)染OCM一1細(xì)胞后,HIF一1QmRNA和蛋白表達(dá)量均下凋;EPO的mRNA和蛋白表達(dá)量也均下調(diào)。:3.EP0作為HIF一1Q的下游靶基因,其表達(dá)受HIF一1a調(diào)控。lUlIlUlUlIIIIIIIIIY2338237碩士研究生:孟凡君(眼科學(xué))導(dǎo)師:周占宇副教授關(guān)鍵詞:脈絡(luò)膜黑色素瘤;缺氧誘導(dǎo)因子一1Q;促紅細(xì)胞生成素;小片段干擾RNA;RNA干擾EffectofSilencingHIF.1僅bysiRNAonExpressionofErythropoietinInMalignantM
5、elanomaoftheChoroidCellAbstractunderHypoxiaObjectives:Toinvestigatetheexpressionofhypoxiainduciblefactor一1aanderythropoietininmalignantmelanomaofthechoroidcelllineOCM.1underhypoxiabymeansoftransferringsmallinterferenceRNAwithcationicliposome.Inordertoobservetheef
6、fectofHIF一1O【inmalignantmelanomaofthechoroid.Methods:OCM一1cellofnormoxicgroupwereculturedonceiicuItureflask1nhighconcentrationofglucoseDMEMcontaining10%FBS,lOOU/mlpenicillin.100gg/mlstreptomycin,andthecellsaremaintainedat37℃inahumidified5%C02incubator.Wetookthece
7、llingoodconditionforexperiment.WeaddedchemicalhypoxiainducerCOCl2intonutrientmediumattheconcentrationof1009mol/LtosimulatehypoxiamicroenvironmentinsidetumortocuIturehypoxicgroupcell·Andhypoxicgroupcellweredividedintofivegroups:simplehypoxicgroup,interferencegroup
8、,negativecontrolgroup,positivecontrolgroup,andliposomegroup.WedesignedandsynthesisedsiRNA(siRNA+negativecontrol+positivecontr01),thetargetsequencesoftheHIF-laa