鼠李糖乳杆菌胞外囊泡对MC3T3-E1细胞生物学特性的影响OA

Objective·To observe the effects of extracellular vesicles derived from Lactobacillus rhamnosus GG(L.GG-EVs)on the proliferation,migration,and osteogenic differentiation of mouse embryonic osteoblast precursor cell line(MC3T3-E1).Methods·After L.GG-EVs were extracted by ultracentrifugation,the morphology of L.GG-EVs was observed by transmission electron microscope(TEM),and the particle size distribution of L.GG-EVs was measured by ZETAView nanoparticle size tracker.After SDS-PAGE,Coomassie brilliant blue R-250 staining was used to observe the protein expression of L.GG-EVs.The effect of L.GG-EVs(12.5,25,50,and 100 μg/mL)on the viability of MC3T3-E1 cells after 1,3,5,and 7 d of co-culture was evaluated by a cell counting kit-8(CCK-8)assay.The wound-healing assay was used to detect the effect of L.GG-EVs(50 and 100 μg/mL)on the migration ability of MC3T3-E1 cells after 6 and 24 h of co-culture.Alkaline phosphatase(ALP)staining was used to detect the effect of L.GG-EVs(50 and 100 μg/mL)on the ALP expression in MC3T3-E1 cells following 7 d of co-culture.Alizarin red S staining was used to detect the effect of L.GG-EVs(50 and 100 μg/mL)on calcium nodule formation in MC3T3-E1 cells after 21 d of co-culture.Quantitative real-time PCR(qPCR)was used to detect the mRNA expression of osteogenic genes,including Runt-related transcription factor 2(Runx2),osteopontin(Opn),and bone morphogenetic protein 2(Bmp2),in MC3T3-E1 cells after 7 d of co-culture with L.GG-EVs(50 and 100 μg/mL).A rat alveolar bone defect model was constructed and divided into the surgical group(SUR group),the pure hydrogel group[gelatin methacryloyl(GelMA)group],and the hydrogel group loaded with L.GG-EVs(GelMA-L.GG-EVs group).The alveolar bone regeneration was detected by Micro-CT.Results·L.GG-EVs were spherical,double-layered membrane vesicles,with diameters ranging from 20 to 500 nm and an average diameter of approximately 161 nm,showing a normal distribution.A protein band was observed at a relative molecular mass of 42 000.CCK-8 assay showed that compared with the CON group,L.GG-EVs at 50 and 100 μg/mL significantly promoted the proliferation of MC3T3-E1 cells after co-culture for 3,5,and 7 d(all P<0.05).The wound-healing assay indicated that L.GG-EVs(50 and 100 μg/mL)accelerated the migration of MC3T3-E1 cells compared with the Ctrl group(all P<0.05).After 7 d of co-culture,L.GG-EVs(50 and 100 μg/mL)markedly intensified ALP staining in MC3T3-E1 cells(both P<0.05).After 21 d of co-culture,L.GG-EVs(50 and 100 μg/mL)significantly increased calcium nodule formation in MC3T3-E1 cells(both P=0.001).After 7 d of co-culture,50 μg/mL L.GG-EVs up-regulated the expression of Opn and Bmp2 mRNA(both P<0.05),and 100 μg/mL L.GG-EVs up-regulated the expression of Runx2,Opn,and Bmp2 mRNA(all P<0.05).In the rat alveolar bone defect model,L.GG-EVs significantly promoted alveolar bone regeneration(all P<0.05).Conclusion·L.GG-EVs can promote the proliferation,migration,and osteogenic differentiation of MC3T3-E1 cells,and have the potential to facilitate bone tissue regeneration.