慢病毒介导shrna沉默fg12基因对心肌微血管内皮细胞增殖、迁移的影响与其机制的的研究

《慢病毒介导shrna沉默fg12基因对心肌微血管内皮细胞增殖、迁移的影响与其机制的的研究》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、摘要(3)酶切及DNA测序表明，fgl2shRNA慢病毒表达载体构建成功。包装慢病毒，测得浓缩病毒滴度为1幸109TU／ml，．转染fgl2shRNA慢病毒96h后，70．80％的MMVECs表达GFP，qRT．PCR结果显示，与实验A组与B组比较，实验C组的龟12表达明显下调，Angl、An92、Tie2、AKT2明显上调，MMVECs的增殖能力与迁移能力增强。具有统计学差异(P

2、ie2、AKT2明显上调；fgl2shRNA慢病毒转染MMVECs，MMVECs的增殖能力与迁移能力增强，可能通过上调Angl和An92的表达来促进血管生成，龟12基因通过RNA干扰为治疗血管新生障碍性疾病提供了新的靶点。关键词：纤维介素2(f912)；慢病毒；短发夹RNA；-t∑,Ct微血管内皮(MMVECs)1IIAbstractAIM：ABSTRACTToisolateandcultureMyocardialMicrovascularEndothelialCellsprimarily,andidentifyitbyimmunofluorescence．To

3、constructalentiviralRNAinterferencedvectorofhumanfibrinogen—likeprotein2(hfgl2)gene．MMVECsweretransfecttedbylentiviralvector．Toinvestigatetheroleoff912geneintheproliferationandMigrationofMMVECs．METHODS：(1)IsolatedandculturedMyocardialMicrovascularEndothelialCellsprimarily,andidentifi

4、edit．Inthisexperiment，wegothearttissuesfromhealthyandcleanSprague—Dawley(SD)ratsofsevendaysold．Weremovedtheheart，washedtheblood，cutatriumandrightventricleoff,kepttheleftventricle，peeledofftheendocardialandepicardial．Digestedhearttissue晰thTrypsinandCollagenaseII，Tllecellcultureswerepu

5、rifiedbydifferentspeedsofattachment．RemovedculturemediumandpassagedMMVECswhenendothelialcellshadgrowninprimarymonolayerfusion．Second—generationMMVECswereidentifiedasendothelialCellsbyfactorVIIIandCD31relatedantigen，whichwereimportantmarkersof．MMVECs(2)111enucleotidesequencesofhfgl2(N

6、M_006682)werefoundintheOeneBank．．Thefourtatgetsequencesoff912genecouldbeeffectivelyscilencedwithRNAinterferencewereconfirmed．ThecDNAcontainingbothsenseandantisenseOligoDNAfragementsoftargetsequencesweredesignedandclonedintothepGCSIL—GFPvectorwhichwasdigestedbyAgeI／EcoRI．Theobtainedle

7、ntiviralvectorcontainingFgl2shRNAwasconfirmedbydigestionandsequencing．UsinglentiviralvectorpHelper1．0、pHelper2．0、pGC·LV龟12totransfect293Tcells，thenthelentiviruswerecollected．ThetiterofviruswastestedaccordingtotheexpressionlevelofGFEIVAbstract(3)Thisexperimentincludedthreegroups：group

8、A，groupBandg