摘要
目的研究DNA甲基化转移酶3B(DNA methyltransferase 3B,DNMT3B)调控人脐带间质干细胞骨生成的作用。方法利用RNA干扰法获得两株DNMT3B基因低表达的人脐带间质干细胞(Human Umbilical Mesenchymal Stem Cells,hUMSCs)。在使用qPCR和免疫组织化学法确定DNMT3B表达缺失后,对hUMSCs进行骨生成定向诱导分化。并利用免疫组织化学法和茜素红染色评价突变型和野生型hUMSCs成骨能力差异。结果由RNA干扰获得的两株突变型hUMSCs中两株突变型中DNMT3B表达量分别下降至野生型hUMSCs的12.67±0.45%和35.60±0.76%(P<0.05)。而在DNMT3B突变型hUMSCs中间质干细胞标记物CD73和CD90基因mRNA的表达水平也分别下降(P<0.05)。CD73水平分别是野生型细胞的15.83±0.44%和16.60±0.37%(P<0.05);CD90的水平分别是野生型细胞的35.23±0.30%和58.64±0.71%(P<0.05)。在骨生成过程中,免疫组化染色后镜下比较显示DNMT3B突变型的细胞表达Osteocalcin较正常细胞低,qPCR结果表明RUNX2,IBSP和ALPL基因的mRNA表达量水平较正常细胞均明显下降(P<0.01),其中shD3B-1和shD3B-2的RUNX2基因表达水平分别为正常细胞的55.09±0.53%和53.77±0.47%(P<0.01);IBSP基因表达水平分别为正常细胞的53.21±0.81%和41.75±0.44%(P<0.01);ALPL基因表达水平分别为正常细胞的15.55±0.10%和17.86±24%(P<0.01)。同时茜素红染色检测也显示其成骨后细胞成骨功能减弱。结论 DNMT3B突变或缺失会导致hUMSCs骨生成能力下降。
Objective To elucidate the roles of DNMT3B in the osteogenesis of hUMSCs.Methods Knockdown DNMT3B in hUMSCs was gained via RNA interference technology.After confirming the decrease of DNMT3B in mutant hUMSCs by immunostaining and qPCR, osteogenesis differentiation was carried out.To identify the phenotype of osteogenesis in both bone formation ability and function of bone,immunostaining,qPCR and functional test was preformed,compared to widetype hUMSCs.Results The mutant hUMSCs showed lower DNMT3B expression level by qPCR.Compared to widetype cells,the DNMT3B expression level were 12.67±0.45% and 35.60±0.76%in two mutant hUMSCs (P<0.05).Furthermore,the expression level of CD73 and CD90 that were two mesenchymal specific markers were 15.83±0.44% and 16.60±0.37% of widetype hUMSCs (P<0.05).Immunostaining showed Osteocalcin positive cell was significant less in DNMT3B mutant cells.qPCR demonstrated that RUNX2,IBSP and ALPL mRNA level were decreased in mutant cells (P<0.01).Moreover,alizarin red staining performed that functional impairment lacking of DNMT3B in hUMSCs.Conclusion The osteogenesis differentiation of hUMSCs is impaired in the absence of DNMT3B.
出处
《外科研究与新技术》
2012年第2期103-107,共5页
Surgical Research and New Technique
基金
国家国际科技合作计划资助项目(2011DFB30010)
