骨髓间充质干细胞培养液对牙周组织再生的影响

Conditioned media of bone marrow mesenchymal stem cells enhanced periodontal tissue regeneration

目的观察大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)培养液对牙周组织再生的影响。方法原代培养SD大鼠BMMSCs,取细胞培养液(conditioned media,CM)。采用ELISA检测培养液里与成骨相关的IGF-1、VEGF、PDGF-BB、BMP-2的含量;利用BMMSCs培养液对牙周膜细胞(periodontal ligament cells,PDLCs)进行培养后,RT-PCR法检测PDLCs矿化相关基因核心结合因子2(Runx2)、骨桥素(OPN)、骨钙素(OCN)的变化;Western印迹法检测Runx2、OPN、OCN蛋白的表达变化。将BMMSCs培养液及种子细胞分别制备成凝胶,在大鼠左下第一磨牙颊侧及根分叉处制备牙周缺损,放入制备好的凝胶。分别在术后第4、8周,处死大鼠,取出左侧下颌骨,进行M icro-CT扫描及组织学分析。结果 BM M SCs培养液中含有大量IGF-1和VEGF;RT-PCR和Western印迹法均能检测到牙周膜细胞OPN、OCN、Runx2的表达,且利用BMMSCs培养液培养的PDLCs表达高于常规培养的PDLCs(P<0.05)。8周时,BMMSCs培养液凝胶组相比于对照组生成了更多的牙槽骨(P<0.05)。结论同等条件下,BMMSCs培养液比种子细胞能更好地修复缺失的牙周组织。

Objective To investigate the effects of conditioned media(CM)of rat bone marrow mesenchymal stem cells(BMMSCs)on periodontal tissue regeneration.Methods Primary bone marrow stromal stem cells were isolated and cultured in vitro.The IGF-1,VEGF,PDGF-BB and BMP-2 levels in conditioned media of BMMSCs were measured by ELISA.The periodontal ligament cells(PDLCs)were cultured with conditioned media,the expression of Runx2,OCN OPN mRNA and protein was detected by RT-PCR and Western blotting,respectively.The gels containing BMMSCs(control group)or their conditioned(test group)were implanted in prepared periodontal defects of rat mandible.After 4 and 8 weeks,the implant sections were examined by micro-CT and histological analysis.Results The conditioned media contains large amounts of IGF-1 and VEGF.The expression levels of Runx2,OPN,OCN mRNAs and protein were up-regulated(P<0.05).After 8 weeks,more bones were recovered in the conditioned media group compared with the control group(P<0.05).Conclusion Conditioned media of BMMSCs can repair the periodontal defect better than seed cells.