简介:LFA-1andMac-1,twoβ2integrinmembersconstitutivelyexpressedonneutrophils,mediateleukocyterecruitmentcascadebybindingtothesameligandofICAM-1.TheslowrollingandfirmadhesionofleukocytesrelyonLFA-1whilethecellcrawlingisdependentonMac-1.Wehypothesizedthattheirdistinctroleswerelikelyattributedtothedifferencesinthebindingkineticsorinthediverseresponsesofoutside-inandinside-outsignaling.Inthisstudy,wecomparedtheICAM-1bindingfeaturesbetweensolubleormembrane-expressedLFA-1andMac-1withdifferentaffinityconformationsusingopticaltraptechnique.Ourdataindicatethattheaffinityup-regulationfromwidetype(WT)tohighaffinity(HA)isoff-ratedependentforLFA-1buton-ratedependentforMac-1.Thestructuralbasesofthisnewfindingwerefoundtobeconsistentwithourprevioussimulations.Theseresultsfurtheredourunderstandingontheirfunctiondifferencesundershearflow.
简介:Objective:Toachieveanoptimizedmethodforsolubleexpressionofhumancarboxylesterase1(hCE-1)inescherichiacoilandpurificationbyNi2+-NTAagaroseaffinitychromatography,togetimprovedproteinyieldandpurityforfurtherdevelopmentofhepatocellularcarcinoma(HCC)diagnosisELISAkits.Methods:ThebestantigenepitopesofhCE1werepredictedbycomparingsecondarystructure,flexibleregions,hydrophilicity,antigenicindexsurfaceprobabilityofresidues.Afterwards,pET-42a(+)withaHis-tagandaGST-tagwasappliedtoformrecombinantplasmidpET-42a(+)/hCE1,whichfacilitatedpurificationwhenusingNi2+-NTAagaroseaffinitychromatography.ProteinqualitywasmeasuredbySDS-PAGEandBCAproteinassay.Western-blotidentificationwasalsoperformedtoensurethecorrectexpressionofhCE1protein.Results:Theresiduesfrom500to567nearC-terminalofhCE1proteinwereconsideredthebestepitopeswhichexhibitedhighhydrophilicityandhighsurfaceprobabilityandrelativelyflexiblesecondarystructureandlowhomologycomparedwithhCE2andhCE3.His-hCE1500-567fusionproteinwasachievedbyIPTG-inductedexpressionwithanexpectedmassof42kDa.Afterpurification,thefinalproductwasspeciallyidentified,whichreachedover95%purityandmorethan10mg/Lofmicrobialculture.InWesternblot,thepurifiedfusionproteinwasrecognizedbyanti-hCE1monoclonalantibody,alongwithprevioussequencingvalidation,whichdemonstratedthecorrectpreparationofsolublehCE1protein.Conclusion:ThisisanefficaciousandaffordablestrategytogeneratefusionhCE1ofhighqualityinEcoli,whichfacilitatespreparationofhCE1monoclonalantibodyandfurtherHCCdiagnosisresearch.
简介:目的:筛选ATP结合盒E1(ABCE1)基因的相关调节miRNA,为诊治肺癌提供新思路。方法选取20例非小细胞肺癌患者,其中男性13例,女性7例;年龄45~73岁,平均年龄62.9岁。鳞癌11例,腺癌9例。应用生物信息学预测ABCE1基因上游的miRNA,通过实时定量聚合酶链反应(RT-Q-PCR)及免疫组织化学方法,对标本非小细胞癌组织和癌旁组织进行检测,并进行统计学分析,从中筛选出目的miRNA。结果生物信息软件预测7个最有可能调节ABCE1基因的miRNA,分别为miR-29a/b/c、miR-135a/b、miR-203及miR-141;其中miR-29a/b/c、miR-135a、miR-203的表达在癌组织内较癌旁组织都有不同程度的降低,以miR-135a、miR-29c差异最为明显,与之对应ABCE1在相同的肺癌组织内表达上调(P〈0.05);仅miR-135a与ABCE1在上述肺癌患者内表达呈现负性相关(r=-0.665,P=0.001)。结论在非小细胞肺癌内,很有可能是miR-135a负性调节ABCE1基因,两者结合可能成为诊治肺癌的新靶点。
简介:Objective:ThispaperistoexploreamethodoftransferringhumanSDF-1anditsmutantSDF-1/54intrakinegeneintoCOS-7cellsfordeterminingtheirexpressionandsubcelluarlocalizationofthefusionprotein.Thiscouldofferfeasibilityforinhibitingthemetastasisofmalignanttumorsbyphonotypicknockoutforblockingfunctionalexpressionofreceptoronthecell-surface.Methods:AmplifythetargetgenewithPCRfromtheconstructedplasimidSDF-WT-Gly×4-Dec/PET-30a(+)withaC-terminalretentionsignalfragmentKDEL.AfterthepcDNA3.1/SDF-1/KDEL,pcDNA3.1/SDF-1/54/KDEL,pEGFP/SDF-1/KDELandpEGFP/SDF-1/54/KDELeukaryoticexpressionvectorswereconstructedandtheDNAsequencewasaccurate,theyweretransferredintoCOS-7cellswithliposome.Theexogenousexpressionswereobserved,fusionproteinSDF-1/HisandSDF-1/54/HiswereconfirmedbyWesternblot,andtheSDF-1/EGFPandSDF-1/54/EGFPweredeterminedbyLaserScanningConfocalMicroscopy.Results:Fourexpressionvectorswereconstructedsuccessfully,thefusionproteinSDF-1/KDEL/HisandSDF-1/54KDEL/HisexpressedinCOS-7cells.SubcelluarlocalizationanalysisshowedthatSDF-1/KDEL/EGFPandSDF-1/54/KDEL/EGFPwerelocatedmainlyinendoplasmicreticulum.Conclusion:FourexpressionvectorspcDNA3.1/SDF-1/KDEL,pcDNA3.1/SDF-1/54/KDEL,pEGFP/SDF-1/KDELandpEGFP/SDF-1/54/KDELwereconstructedsuccessfully,whichcouldexpressineukaryoticcellandlocatemainlyintheendoplasmicreticulum.
简介:CHANGESOF6-K-PGF1aRELEASEFROMTHELUMINALSURFACEOFDACRONSEEDEDWITHAUTOLOOUSVENOUSTISSUEFRAGMENTSCHANGESOF6-K-PGF1aRELEASEFROMTH...
简介:为了探讨束缚+温水(36±1)℃应激是否能引起大鼠胃粘膜损伤、是否对延髓和下丘脑Fos蛋白表达有影响,本研究将雄性Wistar大鼠随机分两组:实验组,束缚+温水(36±1)℃应激1h;对照组,室温下单纯束缚应激1h。应激后处死,测直肠温度;取胃,观察胃粘膜损伤程度;取脑,应用免疫组化染色方法观察两组动物延髓、下丘脑神经元的Fos蛋白表达。结果显示:两组动物胃粘膜损伤程度均较轻或基本无胃粘膜损伤,延髓和下丘脑各核团Fos蛋白表达均无显著性差异,直肠温度也无显著性差异。这些结果提示,由于束缚+温水(36±1)℃应激不改变动物的体温,因而不引起延髓和下丘脑控制胃机能的核团神经元活动加强,从而也不诱发急性胃粘膜损伤。
简介:目的p27Kip1是一种细胞周期素依赖激酶抑制物,它抑制G1期的进程并对其进行调节。方法采用乳化-溶剂挥发法制备含p27kip1的纳米粒子,粒度集中分布在243~343nm,平均粒度为288.9nm,粒径呈窄分布,粒度分布指数为0.192。p27Kip1纳米粒子的载基因率为3%。包封效率为86%。p27Kip1基因纳米粒子的体外释放,开始的5d累积释放曲线接近直线,约1周后释放量开始变慢,释放曲线缓慢上升,可平稳维持释放2周以上。用p27kip1基因纳米粒子转染大鼠动脉平滑肌细胞,分为p27Kip1基因纳米粒子组、空白纳米粒子组、对照组,培养48h后收集细胞,流式细胞仪测定p27kip1纳米粒子对细胞周期调控的结果显示转染前细胞G1/G0期比例为92.4%,转染后48h,对照组及空白纳米粒子组G1/G0期比例为64.5%、68.3%,S期为12.4%、10.3%,表明G0/G1→S的过程非常迅速,细胞增殖活跃。而基因组G1/G0期比例为88.3%,S期为7.2%,说明细胞发生G1期阻滞,细胞增殖受到抑制。建立大鼠自体静脉移植模型,随机分成转基因治疗组、空白纳米粒子组、单纯静脉移植组,应用显微...
简介:Objective:Tostudytheeffectofthreedifferentmethodssuchasmedicine,acupunctureandlaseracupunctureonthelevelsofinterleukin2(IL-2),interleukin6(IL-6)andtransforminggrowthfactor-β1(TGF-β1)inrat’sserumwithchronicatrophicgastritis.Methods:9ratswererandomlyselectedfrom60ratsasnormalcontrolgroup,andtheotherratswereusedtoreplicatetheanimalmodelofchronicatrophicgastritiswithcomprehensivemethod,5ratswererandomlyinspectedatthe8thweekintheprocessofcreatingmodelforgastricmucosapathologicalexamination;whengastricmucosashowsCAGsymptomssuchasvariousdegreeofcongestion,bleeding,atrophy,metaplasia,themodelratswererandomlydividedintomodelgroup,medicinegroup,acupuncturegroupandlaseracupuncturegroup.TheratsofmedicinegroupwerechronicallyadministeredwithyanshenjianweicapsuleandKangfuxinLiquid;thegroupsofacupunctureandlaseracupunctureselectedZusanliacupunctureasacupuncturepoints,afterthetreatmentfor14d,bloodwasremoved,doubleantibodysandwichenzymelinkedimmunosorbentassay(ELISA)wasusedtodetectthelevelsofIL-2,IL-6andTGF-β1inserum.Results:Comparedwithnormalgroup,thecontentsofIL-2,IL-6andTGF-β1inserumofmodelratsincreased(P<0.05orP<0.01).Medicine,acupunctureandlaseracupuncturecouldreducethecontentsofIL-2,IL-6andTGF-β1,IL-2inserumofthemedicinegroupandlaseracupuncturegroupwassignificantlydecreased(P<0.05);IL-6inacupuncturegroupandlaseracupuncturegroupweresignificantlydecreased(P<0.05orP<0.01);andthemostsignificantislaseracupuncturegroup(P<0.01).Medicine,acupunctureandlaseracupuncturecouldsignificantlyreduceTGF-β1contentinserum(P<0.05orP<0.01),andtheparticularlysignificantwaslaseracupuncture(P<0.01).Conclusion:Laseracupuncturehasthefunctionofreducingtheinflammatoryresponseandadjustingimmunefunction.ItcaneffectivelyreducetheexpressionlevelofIL-2,IL-6andTGF-β1inserumofratswithchronicat