骨髓间充质干细胞通过其分泌的外泌体促进髓核细胞增殖的实验研究
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摘要
目的:探讨骨髓间充质干细胞(BMSCs)旁分泌外泌体(exosomes)对髓核细胞的影响。方法:提取并培养骨髓间充质干细胞,通过Exo Quick提取细胞培养液中exosomes,用电镜、nanosight、免疫印迹(western blot)进行鉴定。采用细胞增殖计数(CCK-8)检测髓核细胞的增殖。通过RT-PCR法检测髓核细胞中基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶-3(MMP-3)及抑制物蛋白(TIMP-1)的mRNA水平。结果:成功提取大小在200nm以下BMSCs分泌的exosomes颗粒,其表达CD63及TSG101蛋白。BMSCs分泌的exosomes能够明显促髓核细胞的增殖,且受刺激后髓核细胞内MMP-1、MMP-3表达水平下降,而TIMP-1表达水平升高。结论:骨髓间充质干细胞来源exosomes能够促进髓核细胞增殖,降低髓核细胞的老化,缓解椎间盘组织的退化。
Objective: To investigate the effect of paracrine exosomes secreted by bone marrow mesenchymal stem cells( BMSCs) on nucleus pulposus cells. Methods: BMSCs were extracted and cultured. Exo Quick was used to extract exosomes from cell culture medium,and an electron microscope,nanosight,and Western blot were used for identification.CCK-8 assay was used to measure the proliferation of nucleus pulposus cells. RT-PCR was used to measure the mRNA expression of metal matrix proteinase-1( MMP-1),matrix metalloproteinase-3( MMP-3),and tissue inhibitor of metalloproteinase-1( TIMP-1) in nucleus pulposus cells. Results: The particles of exosomes secreted by BMSCs with a size of < 200 nm were successfully extracted,and the expression of CD63 and TSG101 proteins was found in these particles. Exosomes secreted by BMSCs significantly promoted the proliferation of nucleus pulposus cells,and after stimulation,there were significant reductions in the expression of MMP-1 and MMp-3 and a significant increase in the expression of TIMP-1 in nucleus pulposus cells. Conclusion: Exosomes derived from BMSCs can promote the proliferation of nucleus pulposus cells,delay the aging of nucleus pulposus cells,and alleviate the degeneration of the intervertebral disc.
Objective: To investigate the effect of paracrine exosomes secreted by bone marrow mesenchymal stem cells( BMSCs) on nucleus pulposus cells. Methods: BMSCs were extracted and cultured. Exo Quick was used to extract exosomes from cell culture medium,and an electron microscope,nanosight,and Western blot were used for identification.CCK-8 assay was used to measure the proliferation of nucleus pulposus cells. RT-PCR was used to measure the mRNA expression of metal matrix proteinase-1( MMP-1),matrix metalloproteinase-3( MMP-3),and tissue inhibitor of metalloproteinase-1( TIMP-1) in nucleus pulposus cells. Results: The particles of exosomes secreted by BMSCs with a size of < 200 nm were successfully extracted,and the expression of CD63 and TSG101 proteins was found in these particles. Exosomes secreted by BMSCs significantly promoted the proliferation of nucleus pulposus cells,and after stimulation,there were significant reductions in the expression of MMP-1 and MMp-3 and a significant increase in the expression of TIMP-1 in nucleus pulposus cells. Conclusion: Exosomes derived from BMSCs can promote the proliferation of nucleus pulposus cells,delay the aging of nucleus pulposus cells,and alleviate the degeneration of the intervertebral disc.
引文
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[10]Tao Y,Zhou X,Liu D,et al. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells[J]. Biofactors,2016,42(2):212-223.
[11]Barile L,Lionetti V,Cervio E,et al. Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction[J]. Cardiovasc Res,2017,103(4):530-541.
[2]Strassburg S,Hodson NW,Hill PI,et al. Bidirectional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells[J]. PLo S One,2012,7(13):e33739.
[3]Rani S,Ryan AE,Griffin MD,et al. Mesenchymal stem cell-derived extracellular vesicles:toward cell-free therapeutic applications[J]. Molecular Therapy the Journal of the American Society of Gene Therapy,2015,23(5):812-823.
[4]Chen TS,Lai RC,Lee MM,et al. Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs[J]. Nucleic Acids Res,2009,38(1):215-224.
[5]Toh WS,Lai RC,Hui JHP,et al. MSC exosome as a cell-free MSC therapy for cartilage regeneration:implications for osteoarthritis treatment[J]. Seminars in Cell&Developmental Biology:Elsevier,2017(67):56-64.
[6]Vos T,Flaxman AD,Naghavi M,et al. Years lived with disability(YLDs)for 1160 sequelae of 289 diseases and injuries 1990-2010:a systematic analysis for the Global Burden of Disease Study2010[J]. Lancet,2012,380(9859):2163-2196.
[7]Richardson SM,Kalamegam G,Pushparaj PN,et al. Mesenchymal stem cells in regenerative medicine:focus on articular cartilage and intervertebral disc regeneration[J]. Methods,2016(99):69-80.
[8]Strassburg S,Richardson SM,Freemont AJ,et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype[J]. Regen Med,2010,5(5):701-711.
[9]Rani S,Ryan AE,Griffin MD,et al. Mesenchymal stem cell-derived extracellular vesicles:toward cell-free therapeutic applications[J]. Mol Ther,2015(23):812-823.
[10]Tao Y,Zhou X,Liu D,et al. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells[J]. Biofactors,2016,42(2):212-223.
[11]Barile L,Lionetti V,Cervio E,et al. Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction[J]. Cardiovasc Res,2017,103(4):530-541.