人pre-mir-15a真核表达载体的构建及其对raji细胞生长的抑制作用

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1、编号：刊用形式：人pre-miR-15a真核表达载体的构建及其对Raji细胞生长的抑制作用谌琴，何冬梅(暨南大学医学院血液病研究所,广州510632)摘要：目的构建人pre-miR-15a真核表达载体，并研究其对Raji细胞的生长抑制作用。方法将pre-miR-15a与目标载体(PGCSIL-GFP)定向连接，转化细菌感受态细胞，PCR鉴定阳性克隆，并测序。利用脂质体法将该载体转染Raji细胞，实验分为空白对照组、阴性对照质粒组和pre-miR-15a组。RT-PCR检测Bcl-2mRNA表达，间接免疫荧光法检测B

2、cl-2蛋白表达，台盼蓝细胞计数法检测细胞增殖活性。结果PCR阳性克隆鉴定及测序结果均与目的序列一致。倒置荧光显微镜下见绿色荧光表达；RT-PCR法示各组间Bcl-2mRNA表达差异无显著性（P＞0.05）；间接免疫荧光法示pre-miR-15a组的Bcl-2蛋白表达量较空白对照组和阴性对照质粒组显著降低（P﹤0.05）；台盼蓝拒染法示pre-miR-15a组Raji细胞生长受抑。结论本实验成功构建了per-miR-15a真核表达载体，且pre-miR-15a可以抑制Raji细胞生长。关键词：pre-miR-15a

3、；Raji细胞；淋巴瘤中图法分类号：文献标识码：AConstructionofEukaryoticExpressionVectorofpre-miR-15aandtheinhibitoryeffectofpre-miR-15atoRajicellsCHENQin,HEDong-mei(InstituteofHematology,MedicalCollge,JinanUniversity,Guangzhou510632,China)Abstract：objectiveToconstructeukaryoticexpr

4、essionvectorofpre-miR-15a,andtostudytheinhibitoryeffectofpre-miR-15atoRajicells.MethodsThepGCSIL-GFPvectorwhichwasLineatingwithpre-miR-15anucleotideswastransfectedintothebacterialcompetentcell,andconfirmedbyPCRandsequenceanalysis.TransfectingthisvectorintoRaji

5、cellswitholigofectamine2000,andtheexperimentwasclassedintothreegroups:blank,negativecontrolandpre-miR-15agroup.Semi-quantitativeRT-PCRdetectedtheexpressionlevelofBcl-2mRNA;Bcl-2proteinexpressionwasdetectedbyindirectimmunofluorescence;Thegrowthinhibitoryeffecto

6、fRajicellswasmeasuredbytrypanbluedyeexclusionmethod.ResultstheconsequencesofrecombinantclonesbyPCRandsequencesanalysiswerebothcoinsidedwithtargetsequences.Manygreenfluorescentcellswereobservedunderfluorescentmicroscopy.ThelevelsofBcl-2mRNAateverygrouphadnoobvi

7、ouslydifference.Bcl-2proteinexpressionlevelsobviouslydecreasedatpre-miR-15agroupcomparedwiththeothergroups.Trypanbluedyeexclusionmethodshowedthecellgrowthwasinhibitedat48hand72hpost-transfection.ConclusionWesuccessfullyconstructtheeukaryoticexpressionvectorofp

8、re-miR-15a,andthepre-miR-15acaninhibitthegrowthofRajicells.Keywords：pre-miR-15a;rajicells;lymphoma编号：刊用形式：microRNA(miRNA)是由22个左右核苷酸组成的,基金项目：国务院侨办重点学科建设基金项目(51205002)Supportedbycons