摘要
目的观察骨形成蛋白(bone morphogenetic protein,BMP)7重组腺病毒对大鼠骨髓间充质干细胞(mesenchymal stem cell,MSC)骨向分化的影响。方法构建重组腺病毒载体 pAd-BMP-7,测定其滴度。应用 pad-BMP-7和空白载体 pAdTrack-CMV 分别转染 MSC,检测外源基因转染效率,并应用 RT-PCR、免疫细胞化学手段检测 BMP-7的表达。将 MSC 分为3组,A 组:转染 pad-BMP-7;B 组:转染 pAdTrack-CMV;C 组:转染 pAdTrack-CMV+骨向分化液。观察其矿化结节形成,评价3组细胞骨向分化情况。结果重组腺病毒 pAd-BMP-7的滴度可达2.0×10^(15)pfu/L;外源基因的转染效率为99%,表达时间持续5～7周,3周内表达水平较高。RT-PCR 和免疫细胞化学检测证实了BMP-7在 MSC 中的有效表达。病毒转染后,A 组和 C 组细胞均有矿化结节形成,其中 A 组矿化结节数目显著多于 C 组(P<0.01);B 组无矿化结节形成。结论 BMP-7重组腺病毒可有效转染大鼠骨髓 MSC,并促进其骨向分化,转染的 BMP-7得到了有效表达。
Objective To study the effect of recombinant pAd-BMP-7 on osteogenic differentiation of rat bone marrow mesenchymal stem cells （ MSC ）. Methods Recombinant pAd-bone morphogenetic protein（ BMP）7 was constructed and the titer of recombinant adenovirus was determined, pAd-BMP-7 and pAdTrack-CMV were used to transfect rat MSC. Transfection efficiency was measured by fluorescent microscope and BMP-7 expression was detected by RT-PCR and immunocytochemical analysis. The MSC were then randomly divided into 3 groups： group A received pAd-BMP-7 transfection, group B was transfected with pAdTrack-CMV, and group C received pAdTrack-CMV transfection plus bone supplements. Osteogenic differentiation of MSC was evaluated by examination of mineralization nodes formation. Results The titer of pAd-BMP-7 reached about 2. 0 × 10^15 pfu/L and transfection efficiency of exogenous gene was nearly 99% at day 2. The expression of exogenous gene sustained about 5 to 7 weeks, with a higher level during first 3 weeks. After transfection, transcription of BMP-7 and expression of BMP-7 protein were also verified in MSC. Compared with the negative results in group B, mineralization nodes were formed in both group A and group C. However, group A showed better formation of mineralization nodes than group C （P 〈 0.01）. Conclusions The results of this study indicated that recombinant pAd-BMP-7 can successfully transfect rat MSC and accelerate their osteogenic differentiation. The technique explored in this study provides a unique and valuable gene engineering approach for reconstruction of cranlofacial bone defects.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2007年第4期245-248,共4页
Chinese Journal of Stomatology
基金
国家自然科学基金(30371553)
关键词
干细胞
骨髓祖代细胞
骨形成蛋白
Stem cells
Myeloid progenitor cells
Bone morpbogenetic proteins
