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Abstract：
Oral submucous fibrosis (OSF) is a chronic disease with carcinogenic tendency that poses a non-negligible threat to human health. Exosomes derived from human adipose mesenchymal stem cells (ADSC-Exo) reduces visceral and cutaneous fibroses, but their role in OSF has received little attention. The aim of this study was to investigate the effects of ADSC-Exo on OSF and elucidate the mechanism.
In brief, ADSCs were extracted from adipose tissues and subjected to flow cytometry and induction culture. Fibroblasts were isolated from human buccal mucosa and subjected to immunofluorescence. Myofibroblasts were obtained from fibroblasts induced by arecoline and identified. Immunofluorescence assay confirmed that myofibroblasts could take up ADSC-Exo. The effects of ADSC-Exo on the proliferative and migratory capacities of myofibroblasts were examined using the Cell Counting Kit-8 and scratch assay. Real-time quantitative polymerase chain reaction (qPCR) was performed to evaluate mothers against decapentaplegic homolog 2 (Smad2), Smad3, Smad7, collagen type 1 (Col1), Col3, alpha smooth muscle actin (α-SMA), fibronectin, and vimentin. Western blotting was performed to detect phospho (p)-Smad2, Smad2, p-Smad2/3, Smad2/3, Smad7, Col1, Col3, α-SMA, fibronectin, and vimentin. Furthermore, the dual-luciferase reporter assay was performed to prove that miR-181a-5p in ADSC-Exo directly inhibited the expression of Smad2 mRNA to regulate the transforming growth factor beta (TGF-β) pathway. We also performed qPCR and western blotting to verify the results.
ADSC-Exo could promote the proliferation and migration of myofibroblasts, reduce the expressions of p-smad2, Smad2, p-smad2/3, Smad2/3, Col1, αSMA, fibronectin, and vimentin and elevated the levels of Smad7 and Col3. In addition, miR-181a-5p was highly expressed in ADSC-Exo and bound to the 3\'-untranslated region of Smad2. ADSC-Exo enriched with miR-181a-5p reduced collagen production in myofibroblasts and modulated the TGF-β pathway.
ADSC-Exo promoted the proliferative and migratory capacities of myofibroblasts and inhibited collagen deposition and trans-differentiation of myofibroblasts in vitro. miR-181a-5p in exosomes targets Smad2 to regulate the TGF-β pathway in myofibroblasts. ADSC-Exo perform antifibrotic actions through the miR-181a-5p/Smad2 axis and may be a promising clinical treatment for OSF.
In brief, ADSCs were extracted from adipose tissues and subjected to flow cytometry and induction culture. Fibroblasts were isolated from human buccal mucosa and subjected to immunofluorescence. Myofibroblasts were obtained from fibroblasts induced by arecoline and identified. Immunofluorescence assay confirmed that myofibroblasts could take up ADSC-Exo. The effects of ADSC-Exo on the proliferative and migratory capacities of myofibroblasts were examined using the Cell Counting Kit-8 and scratch assay. Real-time quantitative polymerase chain reaction (qPCR) was performed to evaluate mothers against decapentaplegic homolog 2 (Smad2), Smad3, Smad7, collagen type 1 (Col1), Col3, alpha smooth muscle actin (α-SMA), fibronectin, and vimentin. Western blotting was performed to detect phospho (p)-Smad2, Smad2, p-Smad2/3, Smad2/3, Smad7, Col1, Col3, α-SMA, fibronectin, and vimentin. Furthermore, the dual-luciferase reporter assay was performed to prove that miR-181a-5p in ADSC-Exo directly inhibited the expression of Smad2 mRNA to regulate the transforming growth factor beta (TGF-β) pathway. We also performed qPCR and western blotting to verify the results.
ADSC-Exo could promote the proliferation and migration of myofibroblasts, reduce the expressions of p-smad2, Smad2, p-smad2/3, Smad2/3, Col1, αSMA, fibronectin, and vimentin and elevated the levels of Smad7 and Col3. In addition, miR-181a-5p was highly expressed in ADSC-Exo and bound to the 3\'-untranslated region of Smad2. ADSC-Exo enriched with miR-181a-5p reduced collagen production in myofibroblasts and modulated the TGF-β pathway.
ADSC-Exo promoted the proliferative and migratory capacities of myofibroblasts and inhibited collagen deposition and trans-differentiation of myofibroblasts in vitro. miR-181a-5p in exosomes targets Smad2 to regulate the TGF-β pathway in myofibroblasts. ADSC-Exo perform antifibrotic actions through the miR-181a-5p/Smad2 axis and may be a promising clinical treatment for OSF.
摘要：
背景：口腔粘膜下纤维化（OSF）是一种具有致癌倾向的慢性疾病，对人类健康构成不可忽视的威胁。来自人脂肪间充质干细胞（ADSC-Exo）的外泌体减少内脏和皮肤纤维化，但他们在OSF中的作用很少受到关注。本研究的目的是研究ADSC-Exo对OSF的影响并阐明其机制。
方法：简而言之，从脂肪组织中提取ADSCs并进行流式细胞术和诱导培养。从人颊粘膜分离成纤维细胞并进行免疫荧光。肌成纤维细胞从槟榔碱诱导的成纤维细胞获得并鉴定。免疫荧光实验证实肌成纤维细胞可以摄取ADSC-Exo。使用细胞计数试剂盒-8和划痕测定法检查ADSC-Exo对肌成纤维细胞的增殖和迁移能力的影响。进行了实时定量聚合酶链反应（qPCR）以评估母亲对十一项截瘫同系物2（Smad2）的影响，Smad3，Smad7，1型胶原蛋白（Col1），Col3，α平滑肌肌动蛋白（α-SMA），纤连蛋白,还有波形蛋白.进行蛋白质印迹以检测磷酸（p）-Smad2，Smad2，p-Smad2/3，Smad2/3，Smad7，Col1，Col3，α-SMA，纤连蛋白,还有波形蛋白.此外，通过双荧光素酶报告基因检测,证明ADSC-Exo中的miR-181a-5p直接抑制Smad2mRNA的表达,从而调节转化生长因子β(TGF-β)途径.我们还进行了qPCR和蛋白质印迹以验证结果。
结果：ADSC-Exo能促进肌成纤维细胞的增殖和迁移，减少p-smad2、Smad2、p-smad2/3、Smad2/3、Col1、αSMA的表达式，纤连蛋白,和波形蛋白，并提高Smad7和Col3的水平。此外,miR-181a-5p在ADSC-Exo中高度表达并与Smad2的3'-非翻译区结合。富含miR-181a-5p的ADSC-Exo减少肌成纤维细胞中胶原蛋白的产生并调节TGF-β途径。
结论：ADSC-Exo促进肌成纤维细胞的增殖和迁移能力，抑制肌成纤维细胞的胶原沉积和转分化。外泌体中的miR-181a-5p靶向Smad2以调节肌成纤维细胞中的TGF-β途径。ADSC-Exo通过miR-181a-5p/Smad2轴发挥抗纤维化作用，可能是OSF的有希望的临床治疗方法。
方法：简而言之，从脂肪组织中提取ADSCs并进行流式细胞术和诱导培养。从人颊粘膜分离成纤维细胞并进行免疫荧光。肌成纤维细胞从槟榔碱诱导的成纤维细胞获得并鉴定。免疫荧光实验证实肌成纤维细胞可以摄取ADSC-Exo。使用细胞计数试剂盒-8和划痕测定法检查ADSC-Exo对肌成纤维细胞的增殖和迁移能力的影响。进行了实时定量聚合酶链反应（qPCR）以评估母亲对十一项截瘫同系物2（Smad2）的影响，Smad3，Smad7，1型胶原蛋白（Col1），Col3，α平滑肌肌动蛋白（α-SMA），纤连蛋白,还有波形蛋白.进行蛋白质印迹以检测磷酸（p）-Smad2，Smad2，p-Smad2/3，Smad2/3，Smad7，Col1，Col3，α-SMA，纤连蛋白,还有波形蛋白.此外，通过双荧光素酶报告基因检测,证明ADSC-Exo中的miR-181a-5p直接抑制Smad2mRNA的表达,从而调节转化生长因子β(TGF-β)途径.我们还进行了qPCR和蛋白质印迹以验证结果。
结果：ADSC-Exo能促进肌成纤维细胞的增殖和迁移，减少p-smad2、Smad2、p-smad2/3、Smad2/3、Col1、αSMA的表达式，纤连蛋白,和波形蛋白，并提高Smad7和Col3的水平。此外,miR-181a-5p在ADSC-Exo中高度表达并与Smad2的3'-非翻译区结合。富含miR-181a-5p的ADSC-Exo减少肌成纤维细胞中胶原蛋白的产生并调节TGF-β途径。
结论：ADSC-Exo促进肌成纤维细胞的增殖和迁移能力，抑制肌成纤维细胞的胶原沉积和转分化。外泌体中的miR-181a-5p靶向Smad2以调节肌成纤维细胞中的TGF-β途径。ADSC-Exo通过miR-181a-5p/Smad2轴发挥抗纤维化作用，可能是OSF的有希望的临床治疗方法。
