特立帕肽通过mTORC2通路调控细胞骨架和黏附斑的观察研究
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摘要
目的探讨特立帕肽通过mTORC2通路调控骨髓间充质干细胞的细胞骨架和黏附斑的变化的机制。方法从4~6周大的Sprague-Dawley大鼠中提取骨髓间充质干细胞并培养,使用不同浓度(1、10、20、50 nmol/L)的特立帕肽处理细胞。特立帕肽处理细胞后加入mTORC1抑制剂(20 nmol/L雷帕霉素)或mTORC1/2抑制剂(10μmol/L PP242)后观察现象。细胞迁移采用细胞迁移试验和伤口愈合试验。通过黏附实验观察细胞的黏附能力。蛋白表达的细胞骨架蛋白(β-actin)和黏着斑蛋白(vinculin)用免疫荧光检测。结果在不同浓度特立帕肽的处理下,大鼠的骨髓间充质干细胞相对于对照组呈现出更强的迁移和黏附能力(P <0.05);此外,与1 nmol/L的特立帕肽的浓度相比,10、20、50 nmol/L浓度的特立帕肽均能促进细胞迁移,且处理间差异无统计学意义(P> 0.05);在特立帕肽的刺激下,细胞更大,呈多边形,并且actin应力纤维倾向于有更强的粘连;而加入mTORC1/2抑制剂PP242的骨髓间充质干细胞能显著降低特立帕肽促进迁移和黏附的能力,并且细胞的黏着斑与肌动蛋白末端的骨架纤维较小;加入mTORC1抑制剂雷帕霉素对特立帕肽的抑制作用和则不明显。结论特立帕肽通过激活mTORC2通路调控细胞骨架和黏附斑的变化,进而促进细胞的迁移黏附。
Objective To investigate whether teriparatide regulates cytoskeleton and adhesion plaques of bone marrow mesenchymal stem cells through mTORC2 pathway. Methods Bone marrow mesenchymal stem cells from 4-6 week old Sprague-Dawley rats and were cultured and treated with different concentrations(1 nmol/L,10 nmol/L,20 nmol/L,50 nmol/L of teriparatide then the cells were observed after adding mTORC1 inhibitor(20 nmol/L rapamycin)or mTORC1/2 inhibitor(10 μmol/L PP242). Cell migration assays and wound healing assays were employed for cell migration. The adhesion ability of the cells was observed by adhesion test. Protein-expressed cytoskeletal proteins(β-actin)and focal adhesion proteins(vinculin)were detected by immunofluorescence.Results Rat bone marrow mesenchymal stem cells under different concentrations of teriparatide showed stronger migration and adhesion ability than those in the control group(P < 0.05);in addition,cell migration was promoted with 10,20,and 50 nmol/L of teriparatide when compared to with 1 nm of teriparatide and there was no significant difference between the treatments with different concentrations(P > 0.05). With the stimulation of teriparatide,the cells were larger and polygonal,and actin stress fibers tended to have stronger adhesions;bone marrow mesenchymal stem cells with mTORC1/2 inhibitor PP242 could significantly reduce the ability of teriparatide to promote migration and adhesion. The number of focal adhesion in the cells was reduced and the skeleton fibers at the end of the actin were small. No obvious inhibition of teriparatide by the mTORC1 inhibitor rapamycin wasobserved.Conclusion Teriparatide regulates the changes of cytoskeleton and focal adhesion by activating mTORC2 pathway,thereby promoting cell migration and adhesion.
Objective To investigate whether teriparatide regulates cytoskeleton and adhesion plaques of bone marrow mesenchymal stem cells through mTORC2 pathway. Methods Bone marrow mesenchymal stem cells from 4-6 week old Sprague-Dawley rats and were cultured and treated with different concentrations(1 nmol/L,10 nmol/L,20 nmol/L,50 nmol/L of teriparatide then the cells were observed after adding mTORC1 inhibitor(20 nmol/L rapamycin)or mTORC1/2 inhibitor(10 μmol/L PP242). Cell migration assays and wound healing assays were employed for cell migration. The adhesion ability of the cells was observed by adhesion test. Protein-expressed cytoskeletal proteins(β-actin)and focal adhesion proteins(vinculin)were detected by immunofluorescence.Results Rat bone marrow mesenchymal stem cells under different concentrations of teriparatide showed stronger migration and adhesion ability than those in the control group(P < 0.05);in addition,cell migration was promoted with 10,20,and 50 nmol/L of teriparatide when compared to with 1 nm of teriparatide and there was no significant difference between the treatments with different concentrations(P > 0.05). With the stimulation of teriparatide,the cells were larger and polygonal,and actin stress fibers tended to have stronger adhesions;bone marrow mesenchymal stem cells with mTORC1/2 inhibitor PP242 could significantly reduce the ability of teriparatide to promote migration and adhesion. The number of focal adhesion in the cells was reduced and the skeleton fibers at the end of the actin were small. No obvious inhibition of teriparatide by the mTORC1 inhibitor rapamycin wasobserved.Conclusion Teriparatide regulates the changes of cytoskeleton and focal adhesion by activating mTORC2 pathway,thereby promoting cell migration and adhesion.
引文
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[15]赵胤,彭吾训,张健,等.OPG和RANKL在XACB/LV-bFGF/MSCs移植修复兔股骨头缺损坏死模型中的表达[J].实用医学杂志,2018,34(13):2151-2155
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[17]CHEN B,LIN T,YANG X,et al.Low-magnitude,high-frequency vibration promotes the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells cultured on a hydroxyapatite-coated surface:The direct role of Wnt/beta-catenin signaling pathway activation[J].Int J Mol Med,2016,38(5):1531-1540.
[18]WANG W,CHEN M,GAO Y,et al.P2Y6 regulates cytoskeleton reorganization and cell migration of C2C12 myoblasts via ROCK pathway[J].J Cell Biochem,2018,119(2):1889-1898.
[19]LIU L,LUO Q,SUN J,et al.Cytoskeletal control of nuclear morphology and stiffness are required for OPN-induced bone marrow-derived mesenchymal stem cell migration[J].Biochem Cell Biol,2019.
[20]OMACHI T,ICHIKAWA T,KIMURA Y,et al.Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior[J].Plos One,2017,12(4):e0175324.
[2]HAN H S,JU F,GENG S.In vivo and in vitro effects of PTH1-34 on osteogenic and adipogenic differentiation of human bone marrow-derived mesenchymal stem cells through regulating microRNA-155[J].J Cell Biochem,2018,119(4):3220-3235.
[3]LI H,ZHOU Q,BAI B L,et al.Effects of combined human parathyroid hormone(1-34)and menaquinone-4 treatment on the interface of hydroxyapatite-coated titanium implants in the femur of osteoporotic rats[J].J Bone Miner Metab,2018,36(6):691-699.
[4]ZATI A,SARTI D,MALAGUTI M C,et al.Teriparatide in the treatment of a loose hip prosthesis[J].J Rheumatol,2011,38(4):778-780.
[5]JIANG L,ZHANG W,WEI L,et al.Early effects of parathyroid hormone on vascularized bone regeneration and implant osseointegration in aged rats[J].Biomaterials,2018,179:15-28.
[6]ALMAGRO M I,ROMAN-BLAS J A,BELLIDO M,et al.PTH[1-34]enhances bone response around titanium implants in a rabbit model of osteoporosis[J].Clin Oral Implants Res,2013,24(9):1027-1034.
[7]SATO T,ISHII J,OTA Y,et al.Mammalian target of rapamycin(mTOR)complex 2 regulates filamin A-dependent focal adhesion dynamics and cell migration[J].Genes Cells,2016,21(6):579-593.
[8]HAU A M,LEIVO M Z,GILDER A S,et al.mTORC2 activation is regulated by the urokinase receptor(uPAR)in bladder cancer[J].Cell Signal,2017,29:96-106.
[9]WANG H,SHAO X,HE Q,et al.Quantitative Proteomics Implicates Rictor/mTORC2 in Cell Adhesion[J].J Proteome Res,2018,17(10):3360-3369.
[10]WU D,YU D,WANG X,et al.F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs[J].Cell Prolif,2016,49(6):740-750.
[11]SEN B,XIE Z,CASE N,et al.mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow-derived mesenchymal stem cells[J].J Bone Miner Res,2014,29(1):78-89.
[12]CHEN B,LIN T,YANG X,et al.Intermittent parathyroid hormone(1-34)application regulates c AMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells,via the cAMP/PKA signaling pathway[J].Exp Ther Med,2016,11(6):2399-2406.
[13]WANG X,TANG P,GUO F,et al.RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling[J].Biochim Biophys Acta Gen Subj,2017,1861(1 Pt A):3011-3018.
[14]MA Q L,FANG L,JIANG N,et al.Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces[J].Biomaterials,2018,154:234-247.
[15]赵胤,彭吾训,张健,等.OPG和RANKL在XACB/LV-bFGF/MSCs移植修复兔股骨头缺损坏死模型中的表达[J].实用医学杂志,2018,34(13):2151-2155
[16]SHEYN D,SHAPIRO G,TAWACKOLI W,et al.PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries[J].Mol Ther,2016,24(2):318-330.
[17]CHEN B,LIN T,YANG X,et al.Low-magnitude,high-frequency vibration promotes the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells cultured on a hydroxyapatite-coated surface:The direct role of Wnt/beta-catenin signaling pathway activation[J].Int J Mol Med,2016,38(5):1531-1540.
[18]WANG W,CHEN M,GAO Y,et al.P2Y6 regulates cytoskeleton reorganization and cell migration of C2C12 myoblasts via ROCK pathway[J].J Cell Biochem,2018,119(2):1889-1898.
[19]LIU L,LUO Q,SUN J,et al.Cytoskeletal control of nuclear morphology and stiffness are required for OPN-induced bone marrow-derived mesenchymal stem cell migration[J].Biochem Cell Biol,2019.
[20]OMACHI T,ICHIKAWA T,KIMURA Y,et al.Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior[J].Plos One,2017,12(4):e0175324.
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