牙髓和牙周韧带间充质干细胞成骨能力差异分析研究

The mechanism of the difference of osteogenesis capacity between dental pulp and periodontal ligament stem cells

目的探究牙髓间充质干细胞(dental pulp stem cells,DPSCs)和牙周韧带间充质干细胞(periodontal ligament stem cells,PDLSCs)在成骨能力上的差异机制。方法培养人牙髓干细胞和牙周干细胞(分为DPSCs组和PDLSCs组),比较两种干细胞诱导成骨分化能力的差异,通过实时荧光定量PCR检测成骨分化过程中关键转录因子的转录表达差异。同时,从GEO官网下载牙组织单细胞测序数据(GSE161267_RAW),通过比较干细胞亚群占比差异、调控成骨分化基因表达差异及生物学功能富集等揭示两种组织干细胞成骨分化功能差异的原因。结果成骨诱导分化结果显示,DPSCs组茜素红着色较PDLSCs组更深,运用RT-PCR检测成骨分化关键转录因子骨钙素(osteocalcin,OC)、Runt相关转录因子2(runt-related transcription factor 2,RunX2)、碱性磷酸酶(alkaline phosphatase,ALP)、破骨细胞抑制因子(osteoprotegerin,OPG)和骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)。结果显示,与PDLSCs组相比,DPSCs组Osteocalin、ALP、RunX2、OPG和BMP-2基因转录水平显著升高(**P<0.01,*P<0.05)。单细胞测序数据分析结果显示,牙周和牙髓组织中干细胞构成比例不同,其中牙周组织中MSC2和MSC3亚群占比多,牙髓组织中MSC1亚群占比多;MSC1中调控成骨分化基因表达显著高于MSC2和MSC3。差异基因生物学功能富集结果显示,MSC1亚群主要表现为成骨分化及参与成骨分化的信号通路。结论DPSCs较PDLSCs有更高的成骨分化功能,是由于DPSCs中成骨分化功能强大的细胞亚群含量高于PDLSCs。

Objective To explore the mechanism of the differential osteogenesis capacity between dental pulp stem cells(DPSCs)and periodontal ligament stem cells(PDLSCs).Methods Human pulp stem cells and periodontal stem cells were cultured,the differences in the ability of the two stem cells to induce osteogenic differentiation were compared,and the transcriptional expression differences of key transcription factors in the process of osteogenic differentiation were detected by real-time PCR.Meanwhile,we analyzed single cell sequencing data of human teeth to uncover the characteristic of dental pulp stem cells and periodontal ligament stem cells by using dataset(GSE161267_RAW)downloaded from official website of GEO by comparing the difference in the proportion of stem cell subsets,the expression of genes regulating osteogenic differentiation and the enrichment of biological functions.Results The result of osteogenic induction of differentiation showed that the alizarin red staining in the DPSCs group was darker than that in the PDLSCs group,and the key transcription factors of osteogenic differentiation were detected by RT-PCR,Osteocalcin(OC),Runt-related transcription factor 2(RunX2),alkaline phosphatase(ALP),Osteoprotegerin(OPG)and bone morphogenetic protein-2(BMP-2).The result showed that compared with the PDLSCs group,the transcription levels of Osteocalin,ALP,RunX2,OPG and BMP-2 genes were significantly increased in the DPSCs group(**P<0.01,*P<0.05).The result of single-cell sequencing data analysis showed that the proportion of stem cells in periodontal and pulp tissues was different,with MSC2 and MSC3 subsets accounting for more in periodontal tissues and MSC1 subsets accounting for more in pulp tissues.The expression of genes regulating osteogenic differentiation in MSC1 was significantly higher than that in MSC2 and MSC3.Theresult of biological enrichment of differential genes showed that MSC1 subsets mainly showed osteogenic differentiation and signaling pathways involved in osteogenic differentiation.Conclusion DPSCs have higher osteogenic differentiation function than PDLSCs,because of the cell subsets of DPSCs shows strong osteogenic differentiation potential.