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1、中西醫(yī)結合學報2003年5月第1卷第1期JChinIntegrMed,May2003,Vol.1,No.1·57··技術與方法·成人破骨細胞的體外培養(yǎng)歐陽桂林,楊慶銘,鄧廉夫,許福萍,朱雅萍(上海第二醫(yī)科大學附屬瑞金醫(yī)院骨科,上海市傷骨科研究所,上海200025)[摘要]目的建立成人破骨細胞體外培養(yǎng)的方法,觀察成骨細胞對破骨細胞分化、增殖和功能的影響,為進一步研究補腎中藥防治骨溶解和骨質(zhì)疏松癥提供基礎。材料和方法從成人骨髓中提取單核細胞,以1,25-(OH)2D3作為刺激因子,分3組培養(yǎng):單核細胞與成骨細胞共同培養(yǎng),單核細胞單獨培養(yǎng),
2、單核細胞與成骨細胞條件培養(yǎng)液培養(yǎng)。生物顯微鏡下觀察細胞的分化過程,HE染色和抗酒石酸酸性磷酸酶(Trap)染色方法作組織化學觀察,掃描電鏡(SEM)觀察骨吸收功能。結果在單核細胞與成骨細胞共同培養(yǎng)組,可見單核細胞逐步融合成多核細胞;HE染色和Trap染色可見陽性多核細胞;掃描電鏡觀察可見骨片上有骨吸收陷窩形成。其他兩組則未得到上述結果。結論成人骨髓單核細胞在一定培養(yǎng)條件下能分化成熟為具有典型的破骨細胞表型的多核細胞:Trap染色陽性,具有骨吸收功能。與成骨細胞的直接接觸是破骨細胞分化、增殖和發(fā)揮功能的必不可少的條件。[關鍵詞]細胞培養(yǎng)
3、;破骨細胞;成骨細胞;骨溶解[中圖分類號]R494.26[文獻標識碼]A[文章編號]1672-1977(2003)01-0057-05CultureofhumanosteoclastsOUYANGGui-Lin,YANGQing-Ming,DENGLian-Fu,XUFu-Ping,ZHUYa-Ping(DepartmentofOrthopaedics,RuijinHospital,ShanghaiSecondMedicalUniversity;ShanghaiInstituteofTraumatologyandOrthopeadic
4、s,Shanghai200025,China)[ABSTRACT]ObjectiveToestablishastable,usefulculturesystemforhumanosteoclastsandtoinves-tigatetheeffectofosteoblastsonthedifferentiation,proliferationandactivationofosteoclastssoastopro-videabaseforthestudiesonpreventionandtreatmentofosteolysisando
5、steoporosis.MethodsInthepresenceof1,25-(OH)2D3,monocytesabstractedfromhumanbonemarrowwereculturedinthreegroups:co-cultureofmonocytesandosteoblasts,monocytesalone,monocyteswithconditionalmedia(CM)ofosteoblasts.Differentiationprocessoftheculturedcellswasobservedunderbiolo
6、gicalmicroscope.HEstainingandtartrate-resistantacidphosphatase(Trap)stainingwereemployedtoassaytheculturedcells.Theresorptionpitsonboneslices,onwhichcellswerecultured,wereobservedunderscanningelectronicmicroscope(SEM).ResultsIngroupofco-cultureofmonocytesandosteoblasts,
7、monocytesgraduallyfusedtoformmultinucleatedcells(MNCs),andtheMNCswerealsoindicatedinHEstainingandTrapstaining.TheSEMshowedanumberofresorptionpitsonboneslices.Intheothertwogroups,Trap-posi-tiveMNCswerenotobtaind,andresorptionpitswerenotobservedonboneslices.ConclusionInth
8、iscul-ture,monocytesobtainedfromhumanmarrowfusedtoformmultinucleatedcells(MNCs)thatexpressthemaincharacteristi
