过表达Sema3A促进牙髓干细胞和MC3T3-E1的成骨分化

Overexpression of Sema3A promotes osteogenic differentiation of dental pulp stem cells and MC3T3-E1

背景:Sema3A是一种强有力的骨保护因子。目前,关于Sema3A基因修饰牙髓干细胞(pulp stem cells,DPSCs)在骨再生方面的研究较少。目的:探讨Sema3A修饰的DPSCs(Sema3A-DPSCs)的成骨分化能力,及其对前成骨细胞系MC3T3-E1成骨分化的调控作用。方法:首先,使用慢病毒载体将Sema3A基因转导至DPSCs构建Sema3A-DPSCs,以对照慢病毒处理的DPSCs(Vector-DPSCs)为对照,然后将Sema3A-DPSCs或Vector-DPSCs与前成骨细胞系MC3T3-E1以1∶1及1∶3比例共培养24 h,最后将单独培养的Sema3A-DPSCs、Vector-DPSCs以及二者与MC3T3-E1共培养细胞进行成骨诱导分化,通过碱性磷酸酶染色、茜素红染色、实时定量RT-PCR检测成骨基因表达评价成骨分化能力。结果与结论:(1)Sema3A-DPSCs中Sema3A m RNA及蛋白表达水平显著上调,细胞上清液中分泌型Sema3A水平上调;(2)与Vector-DPSCs相比,Sema3A-DPSCs中成骨相关基因碱性磷酸酶、Runt相关转录因子2、骨钙素、Sp7转录因子的m RNA表达上调,碱性磷酸酶活力增强,矿化结节形成增多;(3)Vector-DPSCs与Sema3A-DPSCs的增殖能力无显著差异;(4)相比于MC3T3-E1/Vector-DPSCs共培养体系,MC3T3-E1/Sema3A-DPSCs共培养体系中MC3T3-E1细胞的成骨相关基因表达上调,总的碱性磷酸酶活性增强并有更多矿化结节形成;(5)结果提示:过表达Sema3A可增强DPSCs的成骨分化能力;在DPSCs中过表达Sema3A可促进DPSCs/MC3T3-E1共培养体系中MC3T3-E1成骨分化。

BACKGROUND:Sema3A is a power secretory osteoprotective factor.However,studies about Sema3A-modified dental pulp stem cells(Sema3A-DPSCs)are rare.OBJECTIVE:To explore the osteogenic differentiation ability of Sema3A-DPSCs and their regulatory effect on the osteogenic differentiation of the pre-osteoblast cell line MC3T3-E1.METHODS:First,Sema3A-DPSCs were constructed using a lentivirus infection system carrying the Sema3A gene.Control lentivirus-treated DPSCs(Vector-DPSCs)were used as controls.Sema3A-DPSCs or Vector-DPSCs were co-cultured with proosteoblast line MC3T3-E1 at the ratio of 1∶1 and 1∶3 for 24 hours.Finally,the Sema3A-DPSCs,Vector-DPSCs and their co-cultured cells with MC3T3-E1 were cultured for osteogenic induction and differentiation.Osteogenic gene expression was detected by alkaline phosphatase staining,alizarin red staining and real-time quantitative RT-PCR to evaluate osteogenic differentiation ability.RESULTS AND CONCLUSION:(1)Sema3A mRNA and protein expression levels in Sema3A-DPSCs were significantly up-regulated.The level of secreted Sema3A in cell supernatant was up-regulated.(2)Compared with the Vector-DPSCs,mRNA expressions of osteogenic genes alkaline phosphatase,Runt-related transcription factor 2,osteocalcin and Sp7 transcription factors in Sema3A-DPSCs were up-regulated;the activity of alkaline phosphatase was enhanced,and the formation of mineralized nodules increased.(3)There were no obvious differences in proliferation between Sema3A-DPSCs and Vector-DPSCs.(4)Compared with MC3T3-E1/Vector-DPSCs co-culture system,the expression of MC3T3-E1 osteogenic genes was up-regulated,and the total alkaline phosphatase activity was enhanced and more mineralized nodules were formed in the MC3T3-E1/Sema3A-DPSCs co-culture system.(5)The results suggest that overexpression of Sema3A can enhance the osteogenic differentiation of DPSCs.Overexpression of Sema3A in DPSCs can promote osteogenic differentiation of MC3T3-E1 in the DPSCs/MC3T3-E1 co-culture system.