sirna沉默hif-1α对缺氧培养的人脉络膜黑色素瘤细胞vegf表达的影响

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1、SiRNA沉默⋯F-1Q对缺氧培养的人脉络膜黑色素瘤细胞VEGF表达的影响摘要目的：研究HIF—lQ小干扰RNA(smallinterferenceRNA，siRNA)转染干扰缺氧诱导的OCM一1细胞前后HIF一1El和VEGF的表达情况，初步探讨HIF一1a在脉络膜黑色素瘤血管生成中的作用。方法：体外培养人脉络膜黑色素瘤细胞(OCM—1)，在培养基中加入CoCl：溶液至终浓度为100umol／L，用于模拟肿瘤内缺氧微环境。生物公司设计并合成siRNA(包括HIF一1QSiRNA、p—actinsiRNA及阴性对照)，以阳离

2、子脂质体(LipofectamineⅢ2000)为载体介导合成的MRNA转染OCM一1细胞。RT—PCR及Western-blot分别在mRNA矛H蛋白水平检测转染前后HIF-IEl并IIVEGF表达的变化。结果：与常氧组相比，单纯缺氧组HIF一1QmRNA表达无明显变化，差异无统计学意义(P>O．05)；HIF一1Q蛋白表达上调，其差异有统计学意义(P

3、的表达均下调，且差异有统计学意义(P

4、GrowthFactorInMalignantMelanomaoftheHumanChoroidCellunderHypoxiaAbstractobjectives：Toinvestigatetheexpressionofhypoxiainduciblefactor·1121(HIF一1O)andvascularendothelialgrowthfactor(VEGF)inmalignantmelanomaofhumanchoroidcell(OCM一1)underhypoxiabeforeandafterOCM一1cell

5、sweretransfectedbysmallinterferenceRNA(siRNA)ofHIF·1Q．TodiscusstheroleofHIF一1Qonangiogenesisinmalignantmelanomaofhumanchoroidcell．Methods：OCM一1cellswereculturedinvitro．ChemicalhypoxiainducerCobaltchloride(CoCl2)wasaddedintonutrientmediumattheconsistencyof100lamol／L

6、tosimulatehypoxiamicroenvironmentinsidetumor．SiRNA(includingHIF一1OsiRNA，p—actinsiRNAandnegativecontr01)weredesignedandsynthetizedbybiotechnologycompany．Cationicliposomes(LipofectamineTM2000)wastakentotransfectsiRNAintoOCM一1cells．Real—TimePCRandWesternblotwereusedto

7、checktheexpressionstatusofHIF·1andVEGFonmRNAandproteinlevelbeforeandaftercelltransfection．Result：Comparedwithnormoxiagroup，therewasnoobviouschangeontheexpressionofHIF一1amRNAinhypoxiagroup．Thedifferencebetweenthemhadnosignificantdifference(P>0．051．TheexpressionofHIF

8、一1aproteininhypoxiagroupwereup—regulatedandtherewasasignificantdifference(P