四种常用的人中性粒细胞分离方法的比较-李金凤

28420088InternationalJournalofPathologyandClinicalMedicineVo.l28No.4Aug.2008:2008-04-15:2008-05-29:(1978-),,,,:,E-mai:[e-mail]:(30400190,30670770);ThisworkwassupportedbytheNationalScienceFoun-dationofChina(30400190,30670770)andtheConstructProgramoftheKeyDisciplineinHunanProvince.李金凤,刘文礼,史小娟,刘伟,汉建忠,万静,罗自强(中南大学湘雅医学院生理学教研室,长沙410078)[]目的:比较Percoll非连续密度梯度离心法Ficol-lHypaque密度梯度离心法裂解红细胞法和Dextran作用下红细胞自然沉降法四种常用的人中性粒细胞分离方法方法:取健康人外周静脉血,分别采用以上四种方法进行中性粒细胞分离,对其细胞纯度回收率存活率进行比较结果:Percoll非连续密度梯度离心法与Ficol-lHypaque密度梯度离心法分离得到的细胞纯度均大于90%,两者间比较无统计学差异(P0.05);裂解红细胞法和Dextran作用下红细胞自然沉降法分离得到的细胞纯度略低于Percoll非连续密度梯度离心法(P0.01)与Ficol-lHypaque密度梯度离心法(P0.05)Dextran作用下红细胞自然沉降法的回收率低于Percoll非连续密度梯度离心法(P0.01)Ficol-lHypaque密度梯度离心法(P0.01)和裂解红细胞法(P0.05);Percoll非连续密度梯度离心法回收的中性粒细胞存活率明显高于Ficol-lHypaque密度梯度离心法(P0.05),裂解红细胞法(P0.01)和Dextran作用下红细胞自然沉降法(P0.01)结论:Percoll非连续密度梯度离心法分离中性粒细胞,纯化程度好,回收率高,是一种简单高效的中性粒细胞分离方法,适于临床和科研中广泛应用[]中性粒细胞;Percoll非连续密度梯度离心法;Ficol-lHypaque密度梯度离心法;裂解红细胞法;Dextran红细胞沉降法[]R331.1[]A[]1673-2588(2008)04-0277-05Comparisonof4popularmethodsforneutrophilisolationfromhumanperipheralbloodLIJin-feng,LIUWen-l,iSHIXiao-juan,LIUwe,iHANJian-zhong,WANJing,LUOZ-iqiang(DepartmentofPhysiology,XiangyaSchoolofMedicine,CentralSouthUniversity,Changsha410078,China)[Abstract]ObjectivePercolldensitygradientcentrifugation,Ficol-lHypaquedensitygradiencentrifugation,redbloodcellscrackingandnaturalerythrocytesedimentationmethodwithDextran,whichare4frequentlyusedmethodsforneutrophilseparationfromhumanperipheralblood,werecom-pared.MethodsRespectivelyusingthe4methodstoseparateneutrophilfromsamehealthyhumanpe-ripheralblood,thepurity,recoveryrate,andcellsurvivalratewerecompared.ResultsAmongthe4separationmethods,theneutrophilpurityfromPercolldensitygradientcentrifugationandFicol-lHypaquedensitygradientcentrifugationweremorethan90%,buttherewasnosignificantdifferencebetweenthem(P0.05);thecellpurityfromredbloodcellscrackingandthenaturalerythrocytesedimentationmeth-odwithDextranwaslowerthanthePercolldensitygradientcentrifugation(P0.01),Ficol-lHypaquedensitygradientcentrifugation(P0.05);theneutrophilrecoveryratefromPercolldensitygradientcen-trifugation,Ficol-lHypaquedensitygradientcentrifugationandredbloodcellscrackingweresignificantlyhigherthanthatfromthenaturalerythrocytesedimentationmethodwithDextran(P0.01,P0.01,and277428P0.05,respectively);CellsurvivalratefromPercolldensitygradientcentrifugationwassignificantlyhigherthanFicol-lHypaquedensitygradientcentrifugation(P0.05),redbloodcellscracking(P0.01),andthenaturalerythrocytesedimentationmethodwithDextran(P0.01).ConclusionPercolldensitygradientcentrifugationprovidesthemostsimpleandefficientapproachforisolationofhu-manbloodneutrophilwiththehighpurityandviability,whichissuitabletoutilizeintheclinicalworkandscientificresearch.[Keywords]neutrophi;lPercol;lFicol-lHypaque;cellscracking;Dextran[IntJPatholClinMed,2008,28(4):0277-05](neutrophil),,,50%~70%,[1](systemicinflammatoryre-sponsesyndrome,SIRS)[2],,,PercollFicol-lHypaqueDextran,,11.1材料,PercollDextranT-500Pharmacia,BD,(BSA),PBS(0.15mol/L,pH7.4)1.2方法1.2.1Percoll非连续密度密度梯度离心法[3-4][4]:Percoll8.77g/L91(V/V),100%Percol,l60%75%Percoll(V/V),60%Percoll1.079,75%Percoll1.090:EDTA2mL,1500r/min15min,(PPP),PBS2mL,2mL75%2mL60%Percoll1mLPPP,,,2000r/min20min60%75%Percoll,PBSPercoll10%RPMI1640200L1.2.2Ficol-lHypaque密度梯度离心法[5]ED-TA2mL,PBS11,11,,2000r/min20min,4:1,2(),3,423,PBS,2784,:28,1000r/min10min,1%BSAPBS,1.077g/mL,2000r/min20min,,PBS,10%RPMI1640200L1.2.3裂解红细胞法分离中性粒细胞[6]EDTA2mL,13(V/V),15min1000r/min5min,1,,1%BSAPBS2mL,4mL60%Percoll,2000r/min20min,1%BSAPBS2,200L1.2.4Dextran作用下红细胞自然沉降法[6]EDTA2mL,400L60g/LDex-tran,3745min,4mL60%Percoll(),2000r/min20min,,3mL15min,PBS5mL,1000r/min5min,1%BSAPBS2,200L1.2.5中性粒细胞的纯度回收率和存活率的测定[5-8]50L,;-,100,;10L,,,200,:=(/)100%,1.3统计学处理(xs),SPSS13.0forwindows,,P0.0522.1四种方法分离得到的中性粒细胞纯度的比较-,,,,,,(1),PercollFicol-lHypaque90%,(P0.05)DextranPercoll(P0.01)Ficol-lHypaque(P0.05)(1),Percoll(2)2.2四种方法分离纯化的中性粒细胞回收率的比较PercollFicol-lHypaque90%,(P0.05),Dextran,Percoll(P0.01)Ficol-lHypaque(P0.01)(P0.05),Percoll(P0.05)(1)2.3四种方法分离所得中性粒细胞存活率的比较90%,Percoll,(P0.01)Dextran(P0.01)Ficol-lHypaque(P0.05)(1)27942814(xs,n=3)Tab.1Comparisonofthepurity,recoveryrate,andcellsurvivalrateofneutrophilsof4kindofseparationmethods(xs,n=3)(%)(%)(%)Percoll92.331.5392.283.0297.171.04Ficol-lHypaque91.331.1590.422.4393.831.04*87.332.52**#88.061.50*92.830.29**Dextran86.671.53**#83.600.81**##92.500.50**Percoll,*P0.05,**P0.01;Ficol-lHypaque,#P0.05,##P0.01;,P0.051PercollFig.1NeutrophilsafterpurificationwithPercolldensitygradientcentrifugation2PercollFig.2LymphcellobtainedfromPercolldensitygradientcentr-ifugation3,,Dextran,[9-10]Dextran1.080~1.085g/mL;1.09~1.095g/mL;1.050~1.066g/mL;1.052~1.077g/mL;1.09~1.11g/mL;1.030~1.060g/mL[11]1.075~1.090,,[12]Ficol-lHypaquePercoll,PercollF-icol-lHypaque,PercollFicol-lHypaqueFicollLPS,,[13]Percoll,[6],Dextran,60%Percoll2804,:28,,,,Dextran,[14],Dextran,,,Percoll[6]PercollFicol-lHypaque,,CD11b,,,(),Percoll,,,,,,1mL,,,[1].[M].5