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ID:31070268
大小:1.93 MB
页数:7页
时间:2019-01-06
《过表达高尔基体α-甘露糖苷酶ⅱ对胃癌细胞株bgc-823增殖的影响》由会员上传分享,免费在线阅读,更多相关内容在工程资料-天天文库。
1、过表达高尔基体α-甘露糖苷酶Ⅱ对胃癌细胞株BGC-823增殖的影响杨紫汐,易永芬,杨雅莹(400016重庆,重庆医科大学病理学教研室,分子医学与肿瘤研究中心)[摘要]目的探讨过表达高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)对人胃癌细胞株BGC-823增殖活性的影响。方法构建真核表达载体EX-E2372-M03,通过脂质体Lipofectamine2000转染至人胃癌细胞株BGC-823,筛选稳定细胞株,用免疫荧光观察转染效率。用RT-PCR法检测转染细胞GMⅡ、c-myc、bcl-2mRNA的表达,用Westernblot法
2、检测转染细胞GMⅡ、c-myc、bcl-2蛋白表达的变化,用MTT法及流式细胞仪检测过表达GMⅡ后对细胞增殖活性的影响。结果GMⅡ真核表达质粒构建成功并成功转染,MTT结果显示过表达GMⅡ后胃癌细胞增殖活性明显高于对照组(P<0.05)。流式检测显示,与对照组相比较,GMⅡ过表达组胃癌细胞G1期细胞明显减少,S期细胞比例明显增加。转染后胃癌细胞GMⅡmRNA和蛋白表达水平明显高于对照组(P<0.05),且可明显上调胃癌细胞BGC-823中c-myc的mRNA和蛋白表达水平(P<0.05),但对bcl-2的调节作用不明显。结论过表达GMⅡ能促进胃癌细胞增殖,
3、可能与上调c-myc的表达有关。[关键词]高尔基体α-甘露糖苷酶Ⅱ;C-myc;Bcl-2;胃癌;增殖[中图法分类号][文献标志码]AEffectofoverexpressedGMⅡonproliferationofgastriccelllineBGC-823YangZixi,YiYongfen,YangYaying(DepartmentofPathology,MolecularMedicineandTumorResearchCenter,ChongqingMedicalUniversity,Chongqing400016,China)[Abstract]
4、ObjectiveToconstructtheeukaryoticexpressionplasmidofhumanGolgiα-mannosidaseⅡ(GMⅡ),identifyitsexpressioningastriccancercelllineBGC-823,andinvestigateitseffectonproliferationofthecells.MethodsTheeukaryoticoverexpressedplasmidvectorEX-E2372-M03wasconstructed,thentransfectedtoBGC-823w
5、ithlipofectamine,andthetransfectionefficiencywasprovedbyFluorescenceMicroscopy.RT-PCRwereusedtodeterminethemRNAexpressionsofGMⅡ、c-myc、bcl-2aftertransfection.FlowcytometryandMTTwereusedtodeterminetheeffectofoverexpressedGMⅡoncellcycledistributionandproliferationactivity.Westernblot
6、wereusedtodetecttheproteinexpressionsofGMⅡ、c-mycandbcl-2aftertransfection.ResultsTheeukaryoticexpressionplasmidofGMⅡwassuccessfullyconstructed,whichwerevalidatedbyDNAsequenceanalysis.RT-PCRanalysisindicatedthatmRNAexpressionofGMⅡandc-mycwasup-regulatedsignificantly(P<0.05)ingastri
7、ccancercelllineBGC-823aftertransfectionwithEX-E2372-M03.Comparetothecontrolgroup,thegrouptransfectedwithEX-E2372-M03showedobviouslyhigherproliferationactivity(P<0.05).FlowcytometricprofilesshowedthatthegroupwithoverexpressedGMⅡledtoadecreaseinthepercentageofcellsinG1phase(P<0.05),
8、andaincreaseinSphase(P<0.05).West
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