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:CHANGESOF6-K-PGF1aRELEASEFROMTHELUMINALSURFACEOFDACRONSEEDEDWITHAUTOLOOUSVENOUSTISSUEFRAGMENTSCHANGESOF6-K-PGF1aRELEASEFROMTH...
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
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: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:目的观察羊体外循环模型中肺损伤的存在,探讨体外循环下肺损伤与水通道蛋白1(AQP-1)基因表达之间的关系。方法健康成年羊16只,雌雄不拘,体质量15-30kg,羊龄8个月。分为脂多糖组和对照组,每组8只。通过建立浅低温体外循环模型,停跳90min,复跳6h,分别比较其血流动力学的指标变化及普通病理组织和超微病理组织的各自特点,测定AQP-1mRNA表达。结果利用体外循环前指标作为自身对照,进行单因素方差分析和Q检验。体外循环缺血再灌注之后,血流动力学较平稳[中心静脉压在体外循环前、心脏复跳前、再灌注至平稳时分别为(5.86±1.12)kPa、(6.52±1.33)kPa、(6.36±1.04)kPa],肺干质量/肺湿质量比值(体外循环前、心脏复跳前、复跳后1h、复跳后3h、复跳后6h分别为0.232±0.025、0.224±0.021、0.187±0.022、0.153±0.018、0.134±0.023)与体外循环时间呈反相关(P〈0.001),血浆总渗透压与体外循环时间呈正相关(P〈0.01)。病理组织证实,复跳之后3-6h可以见到明显的肺间质水肿及血管内皮细胞损伤。肺内AQP-1mRNA表达与体外循环时间呈反相关(体外循环前、心脏复跳前、复跳后1h、复跳后3h、复跳后6h分别为100.0、98.1±24.4、80.2±20.3、78.1±17.7、55.3±16.4),在复跳后3h与6h分别降至术前水平的77.8%和54.6%(P〈0.01)。结论体外循环造成的肺损伤在心脏复跳后3-6h内表现严重,同时AQP-1的表达下降,存在肺血管内皮损伤。
简介:目的总结电视胸腔镜心脏手术体外循环动静脉插管的选择方法及对体外循环灌注过程的影响。方法205例心脏病手术患者,其中男性87例,女性118例;年龄3~46岁,平均年龄16.2岁;体质量13~84kg,平均体质量38.2kg。对其采用胸腔镜建立体外循环,分析股动脉、股静脉及上腔静脉插管型号与患者体质量关系及插管型号对体外循环灌注过程中灌注流量及动脉灌注管阻力的影响。结果114患者例采用股动脉、股静脉插管,91例采用股动脉、股静脉/腔静脉插管建立体外循环。205例患者中有15例在高于2.2L/min流量灌注过程中动脉灌注管阻力高于40kPa(300mmHg),其他患者灌注流量与动脉灌注管阻力均能达到临床要求。结论全胸腔镜体外循环下实施心脏手术选择合适的股动静脉插管可以保证体外循环灌注的有效性与安全性。