兔骨髓间充质干细胞的分离培养及成骨诱导.doc

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1、屮国纽织工程研究»2013,Vol.17»Issue(6):974-979DOI:10.3969/j.issn.2095・4344.2013.06.005骨髓干细胞兔骨髓间充质干细胞的分离培养及成骨诱导Isolation,cultureandosteogenicinductionofrabbitbonemarrowmesenchymalstemcells全文：PDF(499KB)摘要背景：骨髓间充质干细胞具冇多向分化潜能，且可大量体外扩增培养，是巫要的纽织工程种子细胞。但尚无统一的体外培养及定向诱导方法。目的：探讨体外定向诱导兔骨髄间充质干细胞分化为成骨细胞的可行性。方法：应用密度梯度离心法从

2、兔四肢骨屮分离纯化间充质T•细胞，应用密度为1.073g/mL的Percoll分离液,3000r/minx30min离心,区别于相关报道的Ficoll分离液,2000-2500r/minx(20-30)min离心以及全骨髓培养法体外扩增至第3代，分别在普通培养苹(对照组)和成骨诱导培养基(实验组)中培养。结果与结论：成功获得大量高纯度骨攏间充质T•细胞。经成骨诱导后，实验组骨钙索儈量明显高于对照m(P<0.05)o实验纽碱性磷酸酶和钙结节染色阳性，对照纽均阴性。结果表明使川密度梯度离心法可成功建立兔骨髓间充质干细胞的分离培养体系，骨髓间充质干细胞可定向诱导为成骨细胞。关键词：于细胞;骨髄干细胞

3、;骨髓间充质干细胞;'密度梯度离心法;分离培养;成骨诱导;#组织工程;兔；国家自然科学基金;干细胞图片文章;Abstract:BACKGROUND:Bonemarrowmesenchymalstemcellsareimportantseedcellsfortissueengineeringbecauseoftheirmulti-directionaldifferentiationpotentialandpossibilitytobeamplifiedandculturedinvitro.Buttherehavebeennouniformedmethodsofinvitrocultureando

4、rienteddifferentiation.OBJECTIVE:Toinvestigatethefeasiblityofinvitroorienteddifferentationofrabbitbonemarrowmesenchymalstemcellsintoosteoblasts.METHODS:Rabbitbonemarrowmesenchymalstemcellswereisolatedandpurifiedusingdensitygradientcentrifugation(1.073g/mLPercollseparationsolutionforcentrifugationat3

5、000r/minfor30minutes,whichwasdifferentfromFicollseparationsolutionforcentrifugationat2000-2500r/minfor20-30minutesaswellaswholebonemarrowculturemethod).Afterinvitroamplification,passage3bonemarrowmesenchymalstemcellswereculturedwithcommonculturemedium(controlgroup)andosteoblastinductionculturemedium

6、(experimentalgroup).RESULTSANDCONCLUSION:Alargenumberofhighpuritybonemarrowmesenchymalstemcellsweresuccessfullyobtained.Afterosteogenicinduction,thecontentofosteocalcinintheexperimentalgroupwassignificantlyhigherthanthatinthecontrolgroup(P<0.05).Alkalinephosphataseandcalciumtuberclestainingwereposit

7、iveintheexperimentalgroup,buttheywerenegativeinthecontrolgroup・Thesefindingssuggestthatdensitygradientcentrifugationcanbeusedtoisolateandculturerabbitbonemarrowmesenchymalstemcells,andusingthismethod,