Feng Xinhui,Wang Tao,Li Zhuoyang,et al.Osteogenic differentiation characteristics and lncRNA/mRNA regulatory network analysis of periosteal mesenchymal stem cells in patients with congenital pseudarthrosis of the tibia[J].Journal of Clinical Pediatric Surgery,2026,(01):33-42.[doi:10.3760/cma.j.cn101785-202507038]
先天性胫骨假关节患者骨膜间充质干细胞成骨分化特性及lncRNA/mRNA调控网络分析
- Title:
- Osteogenic differentiation characteristics and lncRNA/mRNA regulatory network analysis of periosteal mesenchymal stem cells in patients with congenital pseudarthrosis of the tibia
- Keywords:
- Congenital Pseudarthrosis of the Tibia; Osteogenesis; Osteoclastogenesis; Long Non-Coding RNA
- 摘要:
- 目的 探讨先天性胫骨假关节(congenital pseudarthrosis of the tibia,CPT)患者骨膜来源间充质干细胞(periosteal-derived mesenchymal stromal cells,PDMSCs)的生物学特性及其潜在分子调控机制。方法 本研究为实验性研究。取湖南省儿童医院2020年1月至2020年9月收治的CPT患儿及正常对照人群的胫骨骨膜组织,经酶消化法分离、培养并纯化PDMSCs。通过形态学观察和表面标志物检测验证细胞特征。采用CCK-8法和EdU染色评估PDMSCs的增殖及DNA复制能力。经成骨诱导培养后,进行碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红S染色检测成骨能力,并采用蛋白免疫印迹(Western blot)检测Osterix、Runx2、OCN及OPN等成骨相关蛋白表达。建立PDMSCs与THP-1来源巨噬细胞的共培养体系,诱导破骨分化,采用TRAP(tartrate-resistant acid phosphatase)染色、羟基磷灰石板溶解实验及Western blot检测CTSK、CALCR和ITGβ3等破骨标志物。利用RNA测序(RNA sequencing,RNA-seq)分析CPT与对照PDMSCs的转录组差异,筛选差异表达的mRNAs与lncRNAs,并行GO(Gene Ontology)与KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析。基于差异lncRNA与mRNA的顺式调控关系构建潜在调控网络,筛选成骨相关关键分子组合。结果 CCK-8法结果显示,CPT处理组PDMSCs的细胞活力显著低于对照组(P<0.05)。EdU染色结果显示,CPT处理组PDMSCs的DNA复制水平明显下降(P<0.05)。成骨诱导后,ALP染色强度和茜素红S染色的钙结节形成均明显减弱。Western blot检测结果显示,CPT处理组PDMSCs中Osterix、Runx2、OCN和OPN蛋白表达水平均显著降低(P<0.05)。TRAP染色结果显示,与CPT处理组PDMSCs共培养的巨噬细胞中TRAP阳性细胞比例明显升高。羟基磷灰石板溶解实验表明,共培养体系中溶解活性增强。Western blot结果显示,CPT处理组共培养细胞中CTSK、CALCR和ITGβ3表达水平显著升高(P<0.05)。RNA测序结果显示,CPT处理组PDMSCs中共有822个差异表达mRNAs和194个差异表达lncRNAs,其中mRNAs上调311个,下调511个;lncRNAs上调73个,下调121个。GO与KEGG富集分析结果表明,差异lncRNAs主要富集于色氨酸代谢、类固醇生物合成、鞘脂代谢、谷胱甘肽代谢和初级胆汁酸生物合成等通路。顺式调控分析结果揭示,MIR22HG-WDR81、AC103702-HOXB4、AC004080-HOXA13、lncRNA ATG12-CDO1和lncRNA FAM227B-FGF7等30对lncRNA-mRNA组合具有潜在成骨调控作用。结论 CPT处理组的PDMSCs表现出增殖和成骨分化能力下降、破骨诱导作用增强的特征,差异表达的lncRNA-mRNA调控网络可能在CPT骨形成障碍的发生中发挥关键作用。
- Abstract:
- Objective To investigate the biological characteristics and potential molecular regulatory mechanisms of periosteal-derived mesenchymal stromal cells (PDMSCs) from patients with congenital pseudarthrosis of the tibia (CPT). Methods Tibial periosteal tissues were collected.PDMSCs were isolated,cultured,and purified via enzymatic digestion.Cell morphology and surface markers were used to verify PDMSC characteristics.Cell proliferation and DNA replication were assessed by the CCK-8 assay and EdU staining.After osteogenic induction,alkaline phosphatase (ALP) and alizarin red S staining were performed to evaluate osteogenic capacity,and Western blot was used to detect osteogenesis-related proteins (Osterix,Runx2,OCN,and OPN).A co-culture system of PDMSCs and THP-1-derived macrophages was established to induce osteoclast differentiation.Tartrate-resistant acid phosphatase (TRAP) staining,hydroxyapatite resorption assays,and Western blot for CTSK,CALCR,and ITGβ3 were used to assess osteoclast activity.RNA sequencing (RNA-seq) was conducted to compare transcriptomic profiles between CPT and control PDMSCs,identifying differentially expressed mRNAs and lncRNAs,followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.A cis-regulatory network between lncRNAs and mRNAs was constructed to identify key molecular pairs associated with osteogenesis. Results CCK-8 results showed that cell viability of CPT-derived PDMSCs was significantly lower than that of controls (P<0.05).EdU staining revealed markedly reduced DNA replication in CPT-PDMSCs (P<0.05).After osteogenic induction,both ALP staining intensity and calcium nodule formation (alizarin red S) were weakened.Western blot analysis demonstrated significantly decreased expression of Osterix,Runx2,OCN,and OPN in CPT-PDMSCs (P<0.05).In the co-culture system,TRAP-positive macrophages increased markedly when co-cultured with CPT-PDMSCs.Hydroxyapatite resorption activity was also enhanced.Western blot revealed significantly higher levels of CTSK,CALCR,and ITGβ3 in co-cultures with CPT-PDMSCs (P<0.05).RNA-seq identified 822 differentially expressed mRNAs (311 upregulated,511 downregulated) and 194 differentially expressed lncRNAs (73 upregulated,121 downregulated) in CPT-PDMSCs compared with controls.GO and KEGG enrichment analyses revealed that differential lncRNAs were mainly involved in tryptophan metabolism,steroid biosynthesis,sphingolipid metabolism,glutathione metabolism,and primary bile acid biosynthesis.Cis-regulatory analysis revealed 30 lncRNA-mRNA pairs potentially involved in osteogenic regulation,including MIR22HG-WDR81,AC103702-HOXB4,AC004080-HOXA13,lncRNA ATG12-CDO1,and lncRNA FAM227B-FGF7. Conclusions PDMSCs derived from CPT patients exhibited reduced proliferative and osteogenic differentiation capacity,along with enhanced osteoclast-inducing potential.The differentially expressed lncRNA-mRNA regulatory network may play a crucial role in the impaired osteogenesis observed in CPT.
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备注/Memo
收稿日期:2025-7-17。
基金项目:儿童骨科学湖南省重点实验室(2023TP1019);芙蓉实验室科技项目(2023SK2111)
通讯作者:梅海波,Email:meihaibo@sohu.com