AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖VIP专享VIP免费

生理学报ActaPhysiologicaSinica,April25,2006,58(2):110-115http://www.actaps.com.cn110研究论文Received2005-09-30Accepted2005-11-12ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(No.30470637).*Correspondingauthor.Tel:+86-731-8615806;E-mail:baohewang@126.comAcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖戴国，黄畅，李烨，皮亦华，汪保和*湖南师范大学生命科学学院，长沙410081摘要：N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的四肽因子，对造血干/祖细胞增殖具有抑制作用。本研究采用集落形成实验、甲基偶氮唑盐(MTT)比色法、细胞分裂指数测定等方法,考察了AcSDKP对体外培养的人骨髓间充质干细胞(mesenchymalstemcell,MSC)增殖的影响。结果显示，在AcSDKP浓度为1×10-12mol/L~1×10-9mol/L的培养体系中，人骨髓MSC集落生成率和大小、活力细胞数和分裂指数均降低，最大效应浓度为1×10-11mol/L。以上实验结果表明，在体外培养条件下，一定浓度的AcSDKP对人骨髓MSC的增殖具有抑制作用。关键词：AcSDKP；增殖；抑制；间充质干细胞；骨髓中图分类号：Q253InhibitoryeffectsofAcSDKPonproliferationofhumanbonemarrowmesenchymalstemcellsinvitroDAIGuo,HUANGChang,LIYe,PIYi-Hua,WANGBao-He*CollegeofLifeSciences,HunanNormalUniversity,Changsha410081,ChinaAbstract:ThetetrapeptideN-acetyl-seryl-aspartyl-lysyl-proline(AcSDKP),aphysiologicalregulatorofcellproliferation,hasbeenprincipallyreportedasapotentinhibitoroftheproliferationofhaematopoieticstemcellsandprogenitors.ThepurposeofthisstudywastoinvestigatewhethertheAcSDKPmaydirectlyaffecttheproliferativepotentialofhumanbonemarrowmesenchymalstemcells(MSCs)invitro.WeaddedAcSDKPtotheculturesofhumanbonemarrowmononuclearcellsandmeasuredthenumberandaverageareaofMSCcolonies.MTTcolorimetricassayandmitoticindexdeterminationwerefurtherusedtoexaminetheproliferativestateofthethirdpassageMSCsinsubcultureswithorwithouttheadditionofAcSDKP.Inaddition,weevaluatedwhetherAcSDKPmaykillMSCsbythetrypanbluedyeexclusiontest.Theresultsshowedthatthecolonyformingcapacity,thenumberofviablecellsandthemitoticindexwerereducedinhumanbonemarrowMSCsculturedin1×10-12mol/Lto1×10-9mol/LAcSDKP.Maximuminhibitoryactivityappearedin1×10-11mol/LofAcSDKP.NodifferenceinpercentoflivingcellswasobservedbetweentheMSCsubcultureswithandwithouttheadditionofAcSDKP.Asaresult,AcSDKPwithinacertainrangeofconcentrationshasnegativelyregulatoryeffectsontheproliferationofhumanbonemarrowMSCsinvitro.Keywords:AcSDKP;proliferation;inhibition;mesenchymalstemcell;bonemarrowN-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的肽类因子，由Frindel和Guigon[1]于1977年首次从胎牛骨髓中提取。起初的一系列实验发现，这种四肽因子可通过阻止造血干/祖细胞进入S期，使其停留在G0期，而发挥对细胞增殖的抑制作用[2,3]。后来的研究显示，AcSDKP对非造血细胞如淋巴细胞，肾间质成纤维细胞等也有生长抑制作用[4]。AcSDKP对骨髓间充质干细胞(mesenchymalstemcell,MSC)的影响目前尚未见报111戴国等:AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖道。本研究旨在通过分离、纯化和培养人骨髓MSC，进一步探究AcSDKP对骨髓MSC的生长调控活性。1材料与方法1.1主要试剂DMEM培养基(低糖)、AcSDKP、卡托普利、胰蛋白酶、四甲基偶氮唑盐(MTT)、二甲基亚砜(DMSO)、牛血清白蛋白(BSA)为Sigma产品。胎牛血清(FBS)购于杭州四季青公司。淋巴细胞分离液[(1.077±0.002)g/L]为上海国药集团生化公司产品。荧光素标记小鼠抗人抗体：CD29-PE、CD34-PE、CD45-FITC、CD71-FITC、CD90-FITC购于BD公司。培养瓶、培养皿、塑料培养板为C...