基因转染EphB4对脱落乳牙牙髓干细胞成骨分化的影响  

Effect of transfected EphB4 on osteogenic differentiation in stem cells from human exfoliated deciduous teeth

作　　者：李晓明 袁长永 朱绍跃 刘宗响 王鹏来 

机构地区：[1]徐州医科大学口腔医学院,江苏徐州221004 [2]徐州医科大学附属徐州口腔医院,江苏徐州221002

出　　处：《上海口腔医学》2017年第4期399-403,共5页Shanghai Journal of Stomatology

基　　金：徐州市科技计划项目(Z201628)

摘　　要：目的:建立过表达EphB4的脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED),观察其成骨分化能力的变化。方法:使用酶消化法分离、培养SHED,将第3代SHED分为2组,即病毒介导的EphB4基因转染SHED的实验组和空白荧光载体转染SHED的对照组。通过荧光显微镜、实时荧光定量PCR(real time-PCR)检测转染后SHED细胞中EphB4 mRNA的表达水平,Western印迹法检测转染后EphB4蛋白的表达量。成骨诱导4周后,使用碱性磷酸酶(alkaline phosphatase,ALP)试剂盒及茜素红染色测定转染后SHED的ALP活性改变和成骨能力变化。采用SPSS16.0软件包对数据进行统计学分析。结果:实时荧光定量PCR显示,实验组细胞中EphB4基因表达增高约8倍(P<0.05),Western印迹法显示,实验组细胞中EphB4蛋白表达较对照组显著增高。成骨诱导4周后,ALP检测结果表明,基因转染EphB4,可显著促进SHED的ALP活性(P<0.05),增高约2倍;茜素红染色显示,转染EphB4后,SHED成骨能力显著增强。结论:基因转染EphB4可显著促进SHED的成骨分化能力,为SHED成为骨组织工程中的种子细胞提供了依据。PURPOSE： To evaluate the effect of stem cells from human exfoliated deciduous teeth （SHED） transfected with EphB4 gene in regulating osteogenic differentiation. METHODS： Human dental pulp tissue were harvested from extracted deciduous teeth and digested by collagenase and dispase. The SHEDs were transfected with transgenic （hEphB4- GFP） vector or empty vector （GFP-vector）. Real time-polymerase chain reaction （real time-PCR） analysis and Western blot were used to detect the expression of EphB4 in SHEDs after transfection. EphB4-SHEDs and GFP-SHEDs were subjected to osteogenic induction and assessed by alkaline phosphatase（ALP） assay and Alizarin-red S staining. SPSS 16.0 software package was used for statistical analysis. RESULTS： Real time-PCR revealed that the expression of EphB4 was significantly enhanced in EphB4-SHEDs compared to GFP-SHEDs （P〈0.05）. The expression of EphB4 protein was significantly higher （P〈0.05） in EphB4-SHEDs compared to GFP-SHEDs. ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EphB4-SHEDs. CONCLUSIONS： The results indicate that transgenic expression of EphB4 in SHEDs could promote osteogenic differentiation.

关 键 词：脱落乳牙牙髓干细胞 基因转染 EphB4/ephrinB2 成骨诱导 细胞分化 

分 类 号：R781.3[医药卫生—口腔医学]