[细胞外基质囊泡模拟物的制备和生物学特性]。

Q4 Medicine

中华口腔医学杂志 Pub Date : 2024-07-09 DOI:10.3760/cma.j.cn112144-20231219-00299

X Y Zhang, L Li, S Y Li, J X Liang, F M Chen, Y Yin

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The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. 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引用次数: 0

摘要

研究目的研究机械挤压法制备的细胞外基质囊泡模拟物的特性及其对人牙周韧带干细胞（PDLSC）的细胞活力和成骨分化潜能的影响。方法用胶原酶消化法制备PDLSC细胞外基质囊泡，用机械挤压法模拟细胞衍生的囊泡。获得的细胞外基质囊泡和亲代细胞衍生囊泡模拟物分为四组：在基本培养基中培养 7 天的 PDLSC 基质小泡（PDLSC 基质小泡，MVs）、在基本培养基中培养 7 天的 PDLSC 囊泡模拟物（PDLSC 囊泡模拟物，CVMs）、在成骨诱导培养基中培养 7 天的 PDLSC 所产生的基质小泡（成骨诱导 PDLSC 基质小泡，O-MVs）和在成骨诱导培养基中培养 7 天的 PDLSC 所产生的小泡模拟物（成骨诱导 PDLSC 小泡模拟物，O-CVMs）。通过透射电子显微镜和纳米颗粒跟踪分析观察囊泡的形态和大小。通过免疫荧光检测囊泡的吸收。以PDLSC为对照组，通过细胞活性测定（细胞计数试剂盒-8）检测囊泡对PDLSC活力的影响，通过茜素红染色和Western印迹检测囊泡对PDLSC成骨分化潜能的影响。结果在MVs、O-MVs、CVMs和O-CVMs中观察到的囊泡均为圆形结构（大小为50-250 nm），可被PDLSC吸收而不影响细胞活力。在成骨诱导条件下，与 O-MVs 或 O-CVMs 培养的 PDLSC 比对照组（PDLSC）能产生更多的矿化结节。MVs、O-MVs、CVMs 和 O-CVMs 可促进 PDLSC 中成骨细胞相关蛋白的表达。O-CVMs 组 PDLSC 的成骨相关蛋白表达量明显高于 MVs 组（ALP：1.156±0.170；OPN：1.260±0.293；OCN：1.286±0.302）（P0.05）。与 CMVs 培养的 PDLSC 相比，O-CVMs 培养的 PDLSC 表达更多的 Runt 相关转录因子 2（1.632±0.455 vs 1.176±0.128）和 OPN（1.827±0.627 vs 1.428±0.427）（P0.05）。此外，在用 MVs、O-MVs 和 CVMs 培养的 PDLSC 中，成骨细胞相关蛋白的表达水平无明显差异（P>0.05）。结论机械挤压法制备的囊泡模拟物在形状和大小上与细胞外基质囊泡相似。MVs、O-MVs、CVMs和O-CVMs不会影响PDLSC的细胞活力，并能在一定程度上促进PDLSC的成骨分化潜能。

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[Preparation and biological characteristics of extracellular matrix vesicle mimetics].

Objective: To investigate the characteristics of extracellular matrix vesicle mimetics prepared by mechanical extrusion and their effects on the cell viability and osteogenic differentiation potential of human periodontal ligament stem cells (PDLSC). Methods: PDLSC derived extracellular matrix vesicles were prepared by collagenase digestion, while the cell derived vesicle mimetics were simulated by mechanical extrusion. The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. MVs, O-MVs, CVMs and O-CVMs do not affect the cell viability of PDLSC, and can promote the osteogenic differentiation potential of PDLSC to a certain extent.

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来源期刊

中华口腔医学杂志 Medicine-Medicine (all)

CiteScore

0.90

自引率

0.00%

发文量

9692

期刊介绍： Founded in August 1953, Chinese Journal of Stomatology is a monthly academic journal of stomatology published publicly at home and abroad, sponsored by the Chinese Medical Association and co-sponsored by the Chinese Stomatology Association. It mainly reports the leading scientific research results and clinical diagnosis and treatment experience in the field of oral medicine, as well as the basic theoretical research that has a guiding role in oral clinical practice and is closely combined with oral clinical practice. Chinese Journal of Over the years, Stomatology has been published in Medline, Scopus database, Toxicology Abstracts Database, Chemical Abstracts Database, American Cancer database, Russian Abstracts database, China Core Journal of Science and Technology, Peking University Core Journal, CSCD and other more than 20 important journals at home and abroad Physical medicine database and retrieval system included.