牵张力对小鼠骨髓间充质干细胞成骨分化能力的作用及机制研究-东南大学学报医学版

牵张力对小鼠骨髓间充质干细胞成骨分化能力的作用及机制研究

单位：1. 南方医科大学口腔医学院, 广东广州 510515;
2. 郴州市第一人民医院口腔科, 湖南郴州 423000;
3. 湘南学院附属医院口腔科, 湖南郴州 423000

分类号：R783

出版年·卷·期（页码）：2024·43·第二期（171-176）

摘要：

目的: 探讨牵张力作用对小鼠骨髓间充质干细胞(BMSCs)成骨分化能力的影响及作用机制。方法: 原代分离并培养小鼠BMSCs,并将BMSCs分为对照组、牵张力作用6 h组、牵张力作用12 h组和牵张力作用12 h+雷帕霉素(Rap)组,采用多单元细胞拉伸装置施加动态牵张力进行干预。生化法检测细胞中碱性磷酸酶(ALP)活性;ELISA法检测细胞培养液上清中骨膜蛋白(POSTN)含量;RT-qPCR检测细胞中Runt相关转录因子2(RUNX2)、骨钙素(OCN)、Osterix和骨桥蛋白(OPN) mRNA表达水平;Western blot检测细胞中RUNX2、OCN、Osterix、OPN、POSTN、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR(p-mTOR)蛋白表达水平。结果: 与对照组比较,牵张力作用6 h组和12 h组细胞中ALP活性及RUNX2、Osterix、OCN和OPN mRNA表达水平均显著升高(均P<0.05),上清液POSTN含量、细胞中POSTN蛋白及p-mTOR/mTOR蛋白比值也显著升高(均P<0.05),且牵张力作用12 h组更明显。与牵张力作用12 h组比较,牵张力作用12 h+Rap组细胞中ALP活性和RUNX2、OCN、Osterix、OPN mRNA及蛋白表达水平均显著降低(均P<0.05),而细胞培养液上清中的POSTN含量无明显差异(P>0.05)。结论: 牵张力通过介导POSTN表达调控mTOR信号通路激活,从而促进BMSCs成骨分化。

Objective: To explore the effect and mechanism of tension on osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells(BMSCs). Methods: The primary mouse BMSCs were isolated and cultured, and the BMSCs were divided into control group, tension group for 6 h, 12 h, and 12 h+rapamycin(Rap). Dynamic tension was applied using a multiple-unit cell stretching device for intervention. Biochemical method was used to detect alkaline phosphatase(ALP) activity in cells. ELISA method was used to detect the content of periostin(POSTN) in the supernatant of cell culture medium. RT-qPCR was used to detect the mRNA expression levels of runt related transcription factor 2(RUNX2), osteocalcin(OCN), osterix and osteopontin(OPN) in cells. Western blot was used to detect the protein expression levels of RUNX2, OCN, Osterix, OPN, POSTN, mammalian target of rapamycin(mTOR) and phospho mTOR(p-mTOR) in cells. Results: Compared with the control group, the activity of ALP and the mRNA expression levels of RUNX2, Osterix, OCN and OPN in the 6 h tension and 12 h tension groups were significantly increased(P<0.05), while the POSTN content in supernatant liquid, the expression level of POSTN protein and p-mTOR/mTOR protein ratio were also significantly increased(P<0.05), and the 12 h tension group being more pronounced. Compared with the 12 h tension group, the ALP activity and the expression levels of RUNX2, OCN, Osterix and OPN mRNA and protein in the 12 h tension+Rap group cells were significantly reduced(P<0.05), while the POSTN content in the supernatant of the cell culture medium showed no significant difference(P>0.05). Conclusion: Tension regulates the activation of the mTOR signaling pathway by mediating POSTN expression, thereby promoting osteogenic differentiation in BMSCs.

参考文献：

[1] FU R S,FENG Y L,BERTRAND D,et al.Enhancing the efficiency of distraction osteogenesis through rate-varying distraction:a computational study[J].Int J Mol Sci,2021,22(21):11734.
[2] LIU Z,LIU Q,GUO H B,et al.Overview of physical and pharmacological therapy in enhancing bone regeneration formation during distraction osteogenesis[J].Front Cell Dev Biol,2022,10:837430.
[3] JIN Y Z,LEE J H.Mesenchymal stem cell therapy for bone regeneration[J].Clin Orthop Surg,2018,10(3):271-278.
[4] YANG Y H,PAN Q,ZOU K J,et al.Administration of allogeneic mesenchymal stem cells in lengthening phase accelerates early bone consolidation in rat distraction osteogenesis model[J].Stem Cell Res Ther,2020,11:129.
[5] 蒋校文,黄华庆,陈金勇,等.骨膜蛋白促进兔下颌骨快速牵张成骨的实验研究[J].口腔疾病防治,2019,27(9):551-556.
[6] 陈金勇,蒋校文,杨孝勤,等.小鼠骨髓间充质细胞在牵张力作用下骨膜蛋白的表达及作用[J].北京口腔医学,2018,26(5):260-264.
[7] ROSSELLI-MURAI L K,ALMEIDA L O,ZAGNI C,et al.Periostin responds to mechanical stress and tension by activating the MTOR signaling pathway[J].PLoS One,2013,8(12):e83580.
[8] TANG Y F,SUN Y Q,ZENG J K,et al.Exosomal miR-140-5p inhibits osteogenesis by targeting IGF1R and regulating the mTOR pathway in ossification of the posterior longitudinal ligament[J].J Nanobiotechnol,2022,20:452.
[9] HU X C,LI B E,WU F Z,et al.GPX7 facilitates BMSCs osteoblastogenesis via ER stress and mTOR pathway[J].J Cell Mol Med,2021,25(22):10454-10465.
[10] ZHAO B K,PENG Q,POON E H L,et al.Leonurine promotes the osteoblast differentiation of rat BMSCs by activation of autophagy via the PI3K/Akt/mTOR pathway[J].Front Bioeng Biotechnol,2021,9:615191.
[11] HATEFI S,HATEFI K,ROUX F,et al.Review of automatic continuous distraction osteogenesis devices for mandibular reconstruction applications[J].Biomed Eng Online,2020,19:17.
[12] DIBBS R P,FERRY A M,SARRAMI S M,et al.Distraction osteogenesis:mandible and maxilla[J].Facial Plast Surg,2021,37(6):751-758.
[13] HATEFI S,ALIZARGAR J,ROUX F L,et al.Review of physical stimulation techniques for assisting distraction osteogenesis in maxillofacial reconstruction applications[J].Med Eng Phys,2021,91:28-38.
[14] 蔡杰钦,魏波.骨髓间充质干细胞在牵张成骨中迁移调控机制的研究进展[J].海南医学,2021,32(13):1743-1746.
[15] ORYAN A,KAMALI A,MOSHIRI A,et al.Role of mesenchymal stem cells in bone regenerative medicine:what is the evidence?[J].Cells Tissues Organs,2017,204(2):59-83.
[16] 曹盛楠,师彬,孙国栋,等.生物力学作用对骨髓间充质干细胞增殖和成骨分化影响研究进展[J].山东科学,2021,34(2):65-74.
[17] YONG W K,CHOI J R,CHOI J Y,et al.Recent advances in mechanically loaded human mesenchymal stem cells for bone tissue engineering[J].Int J Mol Sci,2020,21(16):5816.
[18] ZHUANG Y,ZHAO Z Y,CHENG M J,et al.HIF-1α regulates osteogenesis of periosteum-derived stem cells under hypoxia conditions via modulating POSTN expression[J].Front Cell Dev Biol,2022,10:836285.
[19] HAN L Z,GONG S,WANG R Y,et al.Knockdown of POSTN inhibits osteogenic differentiation of mesenchymal stem cells from patients with steroid-induced osteonecrosis[J].Front Cell Dev Biol,2020,8:606289.
[20] LI C R,LI X,WANG X,et al.Periostin mediates oestrogen-induced osteogenic differentiation of bone marrow stromal cells in ovariectomised rats[J].Biomed Res Int,2020,2020:9405909.
[21] ZOU Z L,TAO T,LI H M,et al.mTOR signaling pathway and mTOR inhibitors in cancer:progress and challenges[J].Cell Biosci,2020,10:31.
[22] WANG D,CAI J,ZENG Z B,et al.The interactions between mTOR and NF-κB:a novel mechanism mediating mechanical stretch-stimulated osteoblast differentiation[J].J Cell Physiol,2021,236(6):4592-4603.
[23] ZHAO J,WU J,XU B W,et al.Kaempferol promotes bone formation in part via the mTOR signaling pathway[J].Mol Med Rep,2019,20(6):5197-5207.
[24] GUO Y,FENG L.N6-methyladenosine-mediated upregulation of LINC00520 accelerates breast cancer progression via regulating miR-577/POSTN axis and downstream ILK/AKT/mTOR signaling pathway[J].Arch Biochem Biophys,2022,729:109381.

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