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1、临床医学论文・TGF・B1基因转染BMSCs后复合软骨细胞共培养作者：詹兴旺，李建鑫，卫剑，赵巍，詹兴秀【摘要】H的探讨应用TGF・B1基因转染和软骨细胞共培养后，促进大鼠BMSCsH的基因向软骨细胞的分化效果，为构建新型的软骨种子细胞提供思路。方法6周龄健康雄性Wistar大鼠、扩增、提取质粒pcDNA3.1-TGF-31,酶切、电泳鉴定并测序。实验分组：TGF・01基因转染BMSCs组（A组）、单纯BMSCs与软骨细胞共培养组（B组）、TGF・31转染BMSCs后复合软骨细胞共培养组（C组）、空白BMSCs对照组（D组）、软骨细胞对照组（E组）。结果电泳显示TG
2、F-P1H的基因条带,基因测序与Gene-BankcDNA序列相符。MTT法测定A、B、C、D、E组细胞在490nm波长处的吸光度值，C组与A、B、D、E组间差异有统计学意义（P<0.01）oRT-PCR和Westernblot检测目的基因和蛋白的表达量、TGF・B］表达均以C组最多，A组次之；II型胶原表达以C组最多，A组次结论通过TGF-P1转染同吋复合软骨细胞共培养能有效促进BMSCs向软骨细胞分化，是一个较有前景的发展方向，对组织工程软骨应用于临床具有重要意义。【关键词】BMSCs;共培养;软骨细胞;TGF・31[Abstract]ObjectiveToin
3、vestigatethesecretionoftargetgeneanddifferentiationofBMSCstvansfeetedbyTGF・01andco・cultureandtogetherintochondrocytesandtoprovideanewmethodforculturingseedcelIsincartilagetissueengineering・MethodsTheplasmidspcDNA3.1・TGF・P1wereamplifiedandextracted,thencutbyenzymes,electrophoresedandana
4、lyzeditssequence.BMSCsofWistarratswereseparatedandpurificaledbythedensitygradientcentrifugationandadherentseparation.Themorphologicchangesofprimaryandpassagedcellswereobservedbyinvertedphasecontrastmicroscopeandcellsurfacemarkersweredelectedbyimmunofluorescencemethod.Accordingtothetran
5、sfectsituation,theBMSCsweredividedinto5groups,thetransfectedgroup(GroupA),theco-culturegroup(GroupB),thegroupofTGF・B1andco-culture(GroupC),thegroupofBMSCs(GroupD),thegroupofchondrocytes(GroupE)・Afterbeingtransfected,thecellswereselected,thentheproliferationactivitywastestedbyMTTandexpr
6、essionlevelsweretestedbyRT・PCRandWesternblot.ResultsTheGroupCshowedthatithadagoodprospectandimportantsignificanceofclinicapplicationincartilagetissueengineering.TGF-31genebandsgenesequencingwereconsistentwithGene-BankeDNAsequences.TheabsorbancevaluewithMTTdeterminationofA,bC,D,Egroupof
7、eel1sin490nmwavelengthinCgroupwasstatisticallydifferentwithA,B,D,Egroups(P<0.01).RT-PCRandWesternblotdetectionweredoneforgeneandproteinexpression,TGF-31expressioningroupCweremostAgroupfollowedby;IIcollagenexpressioninCgroupwasmost,Agroupfollowedby.ConclusionByTGF・01transfectingeel1sa
