简介:目的探讨RhoA/ROCK通路特异性阻断剂Y-27632对小鼠骨髓间充质干细胞(bonemarrowmesenchymalstemcells,BMSCs)增殖及成骨分化的影响。方法自小鼠股骨、胫骨骨髓腔分离培养小鼠骨髓单个核细胞,流式细胞仪细胞表面标记检测及细胞成骨、成脂、成软骨三系分化鉴定小鼠骨髓单个核细胞。将第3代小鼠骨髓间充质干细胞随机分为2组,单纯成骨诱导液对照组、Y-27632干预组。分别于细胞培养后1d、2d、3d、4d收集细胞,噻唑蓝(Methylthiazolyldiphenyl-tetrazoliumbromide,MTT)比色法观察BMSCs增殖能力;成骨诱导后7d、14d收集细胞,可见光比色法检测碱性磷酸酶(ALP)变化;成骨诱导后24h收集细胞,westernblot法检测Rho相关卷曲螺旋形成蛋白激酶1(Rhoassociatedcoiled-coil-containingproteinkinases1,ROCK1)的表达。结果流式细胞术细胞表面标志检测及成骨、成脂、成软骨三系分化鉴定均证实我们所获细胞为BMSCs。与对照组相比,Y-27632能促进BMSCs增殖,差异具有统计学意义(P〈0.05);Y-27632能降低ALP表达,差异具有统计学意义(P〈0.05);Y-27632能明显阻断ROCK1的表达,差异具有统计学意义(P〈0.05)。结论Y-27632阻断RhoA/ROCK信号通路可以促进BMSCs增殖,抑制BMSCs成骨分化。
简介:AIM:ToinvestigatetheeffectofY-27632onthesurvivalandneuriteoutgrowthoftheculturedretinalneurocytes.METHODS:Afterthepostnatalday2-3,Sprague-Dawleyretinalneurocyteswereculturedfor48hours,theculturemediawasreplacedwithserum-freemedia(controlgroup)andserum-freemediacontained30μmol/LY-27632(Y-27632group),andthecellswerecontinuallyculturedanother48hours.Theculturedretinalneurocyteswereidentifiedwithanti-neuronspecificenolase(NSE)immunocytochemistry.ThesurvivalstateofthosecellswasestimatedbyMTTassay,andtheneuriteoutgrowthofthosecellswasevaluatedbythecomputerizedimage-analysissystem.RESULTS:Comparedwiththecontrolgroup,theabsorbancevaluesofcellssurvivalinY-27632groupincreased12.90%and33.33%respectivelyafter72and96hoursculture.Y-27632hadnosignificanteffectonthediameterofculturedretinalneurocytes.Comparedwiththecontrolgroup,Y-27632inducedastableimprovementofneuriteoutgrowthofretinalneurocytesafter72and96hoursculture(P=0.001).CONCLUSION:Y-27632couldpromotethesurvivalandneuriteoutgrowthoftheearlypostnatalculturedretinalneurocytes.
简介:背景:Rho激酶抑制剂可以调节细胞骨架重构,激活转录因子,促进细胞增殖和分化。目的:观察Rho激酶抑制剂Y-27632对大鼠骨髓间充质干细胞分裂增殖和细胞周期的影响。方法:采用贴壁细胞培养法体外分离培养SD大鼠骨髓间充质干细胞,并传代。取生长状态良好的第2代骨髓间充质干细胞分组:实验组加入10μmol/LRho激酶抑制剂Y-27632,对照组正常培养。结果与结论:①骨髓间充质干细胞增殖:细胞生长曲线呈“S”型,Rho激酶抑制剂Y-27632作用后,3d左右进入平台期,细胞生长加速,生长曲线上移。②骨髓间充质干细胞的细胞周期:对照组G0/G1期细胞比例为(80.640±5.516)%,S期细胞比例为(13.397±2.511)%;实验组G0/G1期细胞比例为(61.723±8.829)%,S期细胞比例为(29.380±7.630)%,实验组S期细胞比例高于对照组(P〈0.05)。表明Rho激酶抑制剂Y-27632可以促进骨髓间充质干细胞DNA的合成,促进细胞分裂和增殖。
简介:AIM:ToinvestigatetheinterferingeffectofY-27632,aROCK-Iselectiveinhibitor,onthesignaltransductionpathwayoftransforminggrowthfactor-β1(TGF-β1)inocularTenoncapsulefibroblasts(OTFS)invitro.METHODS:AfterOTFSfrompassages4to6invitrowereinducedbyTGF-β1andthentreatedbyY-27632,thechangesoftheOTFScellcycleswereanalyzedviaflowcytometry,andtheproteinsexpressionoftheα-smoothmuscularactin(α-SMA),connectivetissuegrowthfactor(CTGF),collagenIwerecalculatedbyWesternblot.AfterOTFStreatedbythedifferentconcentrationsofY-27632,theexpressionlevelsoftheα-SMA,CTGFandcollagenImRNAwereassayedbyRT-PCR.RESULTS:Y-27632hadnomarkedlyeffectontheOTFScellcycles.AftertreatedbyTGF-β1,OTFSinG1periodsignificantlyincreased.ThecellcyclesdistributionbybothTGF-β1andY-27632hadnoremarkabledifferencefromthatincontrolgroup.Y-27632significantlyinhibitedtheproteinsexpressionsofbothα-SMAandCTGF,whiletosomeextentinhibitedthatofcollagenI.TGF-β1significantlypromotedtheproteinsexpressionsofα-SMA,CTGFandcollagenI.AfterOTFStreatedbybothTGF-β1andY-27632,ofα-SMA,theproteinexpressionwassimilarwiththatincontrolgroup(P=0.066>0.05),buttheproteinexpressionofCTGForcollagenI,respectively,wassignificantlydifferentfromthatincontrolgroup(P=0.000<0.01).Thedifferencesofexpressionsoftheα-SMA,CTGFandcollagenImRNAin30,150,750μmol/LY-27632groupwerestatisticallysignificant,comparedwiththoseincontrolgroup,respectively(α-SMA,P=0.002,0.000,0.000;CTGF,P=0.014,0.002,0.001;collagenI,P=0.003,0.002,0.000).CONCLUSION:BlockingtheRho/ROCKsignalingpathwaybyusingofY-27632couldinhibitthecellularproliferationandtheexpressionofbothCTGFandα-SMAwhateverOTFSinducedbyTGF-β1ornot.Y-27632suppressedtheexpressionofcollagenImRNAwithoutinduction.
简介:目的:通过优化条件培养基用于非牙源性上皮细胞的体外扩增,为进一步开展组织工程牙提供上皮种子细胞来源奠定基础。方法:获取出生后1dC57BL鼠原代口腔黏膜上皮细胞,接种于含有Y27632的上皮条件培养基的培养瓶内。细胞长满后,消化传代,采用条件培养基与3T3饲养层细胞共培养进行扩增并连续传代,观察细胞生长情况及连续传代后的生物学特性,免疫组织化学进行干细胞鉴定。结果:与传统上皮细胞培养方式相比,含Y27632的条件性培养基加3T3饲养层方式培养的上皮细胞生长迅速,细胞呈立方形或多角形,呈铺路石样排列。免疫组织化学染色表明这些干细胞表达CK14,SOX2和LGR5。连续传代至第7代后,细胞形态、生长曲线和干细胞标志没有明显变化。结论:含有Y27632的条件培养基可有效用于体外大量扩增非牙源性上皮细胞,并能保持干细胞生长特性,有望用于组织工程牙的种子细胞来源。
简介:【摘要】在深圳卷烟厂,YJ112卷烟机已作为卷烟卷制环节的主力机型;该机型主要依靠平准器对烟支重量进行控制调节和紧固烟支端部烟丝。通过对YJ112卷烟机平准器主传动法兰部位的改进,采用主传动法兰表面喷涂陶瓷和主传动法兰内孔加装防油垫片等措施,有效减少了平准器漏油的次数;通过安装双重法兰和回油管,进一步降低了平准器漏油风险,加装回油管使得漏油更容易观察;通过实践证明,以上的设计改进是可行的。