Exosomal MATN3 of Urine-Derived Stem Cells Ameliorates Intervertebral Disc Degeneration by Antisenescence Effects and Promotes NPC Proliferation and ECM Synthesis by Activating TGF-β.
Exosomal MATN3 of Urine-Derived Stem Cells Ameliorates Intervertebral Disc Degeneration by Antisenescence Effects and Promotes NPC Proliferation and ECM Synthesis by Activating TGF-β.
复制标题
尿源干细胞外泌体 MATN3 通过抗衰老作用改善椎间盘退变，并通过激活 TGF-β 促进 NPC 增殖和 ECM 合成
DOI：
10.1155/2021/5542241
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发表时间：
2021
影响因子：
--
通讯作者：
Xiang H
中科院分区：
文献类型：
--
作者：
Guo Z;Su W;Zhou R;Zhang G;Yang S;Wu X;Qiu C;Cong W;Shen N;Guo J;Liu C;Yang SY;Xing D;Wang Y;Chen B;Xiang H
腰痛（LBP）是发达国家三大致残原因之一，而椎间盘退变（IDD）是腰痛的主要诱因。IDD过程中，髓核细胞（NPCs）和细胞外基质（ECM）逐渐减少。外泌体是干细胞重要的外分泌介质，可以直接作用于细胞进行组织修复和再生。在本研究中，我们检测了人尿源性干细胞（USC）外泌体（USC-exos）对退变椎间盘的抗衰老、细胞增殖促进和ECM调节作用，并探讨了其潜在机制。采用多能分化和流式细胞术对间充质干细胞（MSC-）特异性表面蛋白标志物进行鉴定。采用超离心的方法从USC-exos培养基中分离出USC-exos，通过透射电镜（TEM）、粒度分析和western blotting （WB）检测外泌体标记蛋白。通过细胞计数试剂盒-8 （CCK-8）、WB和免疫荧光（IF）分析评估USC-exos对NPC增殖和ECM合成的影响。提取正常椎间盘与退变椎间盘的蛋白差异，通过椎间盘组织WB和IF测定MATN3含量的时空变化。筛选了介导USC-exos功能的候选分子，并通过多次实验进行了确认。同时，我们还探讨了usc -exos诱导细胞增殖和调控ECM合成促进NPCs活性的机制。此外，通过计算机断层扫描（CT）、磁共振成像（MRI）和组织学分析，研究了USC-exos对小鼠椎间盘退变（IVD）的改善作用。流式细胞术结果显示，USC表面特异性标志物CD29、CD44和CD73呈阳性，而CD34和CD45呈阴性。此外，USCs显示成骨、脂肪和软骨分化潜能。USC-exos呈杯状，平均直径为49.7±7.3 nm， CD63和TSG101阳性，calnexin阴性。USC-exos可促进NPC增殖和ECM合成。退变椎间盘中基质家族蛋白含量明显降低，其中MATN3的降低最为显著。发现USC-exos富含MATN3蛋白，并且外泌体MATN3是USC-exos诱导的促进IVD大鼠鼻咽癌增殖和ECM合成以及减轻椎间盘退变所必需的。此外，MATN3在USC-exos中的作用被证明是通过激活TGF-β来实现的，TGF-β可以提高SMAD和AKT的磷酸化水平。我们的研究表明，减少的MATN3可以被认为是椎间盘退变的一个特征。USC-exos可能是一种潜在的有效药物，通过转移MATN3蛋白促进NPC增殖和ECM合成，从而减轻椎间盘退变。
Low back pain (LBP) is one of the top three causes of disability in developed countries, and intervertebral disc degeneration (IDD) is a major contributor to LBP. In the process of IDD, there is a gradual decrease in nucleus pulposus cells (NPCs) and extracellular matrix (ECM). Exosomes are important exocrine mediators of stem cells that can act directly on cells for tissue repair and regeneration. In this study, we determined the antisenescence, cell proliferation promotion, and ECM modulation effects of human urine-derived stem cell (USC) exosomes (USC-exos) on degenerated intervertebral discs and explored the underlying mechanism. USCs were identified by multipotent differentiation and flow cytometry for mesenchymal stem cell- (MSC-) specific surface protein markers. USC-exos were isolated from the conditioned medium of USCs by ultracentrifugation and then analyzed by transmission electron microscopy (TEM), particle size analysis, and western blotting (WB) for exosome marker proteins. The effects of USC-exos on NPC proliferation and ECM synthesis were assessed by Cell Counting Kit-8 (CCK-8), WB, and immunofluorescence (IF) analyses. The protein differences between normal and degenerative intervertebral discs were mined, and the temporal and spatial variations in matrilin-3 (MATN3) content were determined by WB and IF in the intervertebral disc tissues. The candidate molecules that mediated the function of USC-exos were screened out and confirmed by multiple assays. Meanwhile, the mechanism underlying the candidate protein in USC-exos-induced cell proliferation and regulation of ECM synthesis promoting the activities of NPCs was explored. In addition, the effects of USC-exos on ameliorating intervertebral disc degeneration (IVD) in mice were examined by assessing computed tomography (CT), magnetic resonance imaging (MRI), and histological analyses. The flow cytometry results showed that USCs were positive for CD29, CD44, and CD73, which are USC surface-specific markers, but negative for CD34 and CD45. In addition, USCs showed osteogenic, adipogenic, and chondrogenic differentiation potential. USC-exos exhibited a cup-shaped morphology, with a mean diameter of 49.7 ± 7.3 nm, and were positive for CD63 and TSG101 and negative for calnexin. USC-exos could promote NPC proliferation and ECM synthesis. The protein content of the matrilin family was significantly reduced in degenerative intervertebral discs, and the decrease in MATN3 was the most significant. USC-exos were found to be rich in MATN3 protein, and exosomal MATN3 was required for USC-exos-induced promotion of NPC proliferation and ECM synthesis, as well as alleviation of intervertebral disc degeneration in IVD rats. In addition, the effects of MATN3 in USC-exos were demonstrated to be achieved by activating TGF-β, which elevated the phosphorylation level of SMAD and AKT. Our study suggests that reduced MATN3 can be considered a characteristic of intervertebral disc degeneration. USC-exos may represent a potentially effective agent for alleviating intervertebral disc degeneration by promoting NPC proliferation and ECM synthesis by transferring the MATN3 protein.
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影响因子：
5.5
作者：
Chai, Chang;Song, Lai-Jun;Li, Ming
通讯作者：
Li, Ming
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影响因子：
5.6
作者：
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通讯作者：
Somanath, Payaningal R.
影响因子：
30.8
作者：
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通讯作者：
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影响因子：
5.3
作者：
Cheng X;Zhang G;Zhang L;Hu Y;Zhang K;Sun X;Zhao C;Li H;Li YM;Zhao J
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影响因子：
7.5
作者：
Feng, Fa-Bo;Qiu, Hai-Yan
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