细胞外基质囊泡模拟物制备及其生物学性能的初步研究

Preparation and biological characteristics of extracellular matrix vesicle mimetics

目的探讨运用机械挤压法制备细胞外基质囊泡模拟物的生物学性能及其对人牙周膜干细胞(PDLSC)细胞活性和成骨分化潜能的影响。方法胶原酶消化法制备PDLSC来源细胞外基质囊泡,机械挤压法制备亲本细胞来源囊泡模拟物;将获取的天然细胞外基质囊泡和亲本细胞来源囊泡模拟物分为4组:基础培养基培养7 d的PDLSC来源基质囊泡(PDLSC matrix vesicles,MVs)和基础培养基培养7 d的PDLSC来源囊泡模拟物(PDLSC vesicle mimetics,CVMs),成骨诱导培养基培养7 d的PDLSC来源基质囊泡(osteogenic-induced PDLSC matrix vesicles,O-MVs)和成骨诱导培养基培养7 d的PDLSC来源囊泡模拟物(osteogenic-induced PDLSC vesicle mimetics,O-CVMs);透射电镜和纳米颗粒示踪分析观察囊泡形态和大小,免疫荧光检测细胞摄取囊泡情况;以PDLSC作为对照组,细胞活性实验检测囊泡对PDLSC细胞活性的影响,茜素红染色和蛋白质印迹法检测囊泡对PDLSC成骨分化潜能的影响。结果MVs、O-MVs、CVMs和O-CVMs均表现为圆形囊泡结构(直径50~250 nm),均能被PDLSC摄取,且不影响PDLSC的细胞活性;成骨诱导条件下,O-MVs和O-CVMs孵育的PDLSC相比于对照组细胞形成更多的矿化结节;MVs、O-MVs、CVMs和O-CVMs均能促进PDLSC表达成骨相关蛋白,O-CVMs孵育的PDLSC表达的碱性磷酸酶(1.571±0.348)、骨桥蛋白(1.827±0.627)和骨钙蛋白(1.798±0.537)表达水平均显著高于MVs孵育的细胞(分别为1.156±0.170、1.260±0.293和1.286±0.302)(均P<0.05),Runt相关转录因子2(1.632±0.455)和骨桥蛋白(1.827±0.627)表达水平均显著高于CMVs孵育的细胞(分别为1.176±0.128和1.428±0.427)(均P<0.05),MVs、O-MVs和CVMs孵育的PDLSC成骨相关蛋白表达水平差异均无统计学意义(P>0.05)。结论机械挤压法制备的囊泡模拟物与天然细胞外基质囊泡的形态、大小类似,不影响PDLSC的细胞活性,并可在一定程度上促进PDLSC的成骨分化潜能。

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.