| Objective: Observe the effects of BMSCs-derived exosomes(BMSCs-Exo)and Runx2 transfected BMSCs-derived exosomes(R-BMSCs-Exo)on chondrocyte proliferation,migration and phenotype maintenance,and explore the role of proliferation,migration and phenotype maintenance Regulatory proteins in the action process,with a view to providing molecular biological evidence for the clinical treatment of patients with extensive cartilage damage.Methods:1.BMSCs extracted in vitro were identified by microscopic observation,osteogenesis,adipogenic induction staining and flow cytometry;chondrocytes isolated in vitro were identified by microscope,and acridine orange and toluidine blue type II collagen immunofluorescence staining were used for identification.2.The transfection efficiency and optimal transfection concentration of Runx2 adenovirus have been determined in previous experiments,which are directly cited in this study.3.After YAP-si RNA screening,the effect concentration and time were explored to determine the best inhibitory effect.4.Normally cultured rabbit BMSCs in vitro,P3 generation cells were divided into simple cell group and Runx2 transfection group for expansion culture.After the end of the culture,the cell supernatant was collected and the exosomes were extracted by ultracentrifugation.5.Normally cultured rabbit knee joint chondrocytes in vitro were taken from P2 seed plates.When the cell density reached 70%,exosomes with different concentrations(0,1,5,10)?g/m L were added,and Col-II,Expressions of phenotype genes and proteins such as SOX9 and Aggrecan(BMSCs-Exo and R-BMSCs-Exo were independently performed during this process and the expression differences between the two peaks were compared).In addition,CCK-8 and Transwell experiments were used to observe the BMSCs-Exo and Cell proliferation and migration levels at the peak of R-BMSCs-Exo.6.Explants were transfected with chondrocytes of P2 generation,and the expression of YAP and its target genes Ankrd1 and CTGF were observed.7.Transfection of exosomes to chondrocytes after silencing YAP with si RNA.Observe the differences in chondrocyte proliferation,migration,and phenotype expression before and after YAP silencing to determine whether the role of exosomes is exerted by YAP.Results:1.Observed by an inverted microscope,the cell morphology was typical long fusiform,spindle-shaped,and the clone growth was arranged in a vortex.After osteogenic induction,a large amount of mineralized nodules can be seen by alizarin red staining;after fat induction,a large number of lipid droplets can be seen by oil red O staining.FCM results showed that high expression of CD44 and CD29,and low expression of CD45,indeed isolated and cultured BMSCs.2.Chondrocytes began to adhere to the wall after 12 h,and began to spread at 24 h.After 5 days,the cells fused into pieces,like a "paving stone".Toluidine blue staining showed that chondrocytes grew in a single layer,with a triangular or polygonal shape,and the cytoplasm was blue-violet;acridine orange fluorescent staining showed that the chondrocyte nucleus showed clear green fluorescence;COL-? immunofluorescence staining showed that the cytoplasm was blue,with nucleus It is red and isolated in vitro as chondrocytes.3.The exosomes were analyzed by NTA,and their particle size distribution was mostly between 30-150 nm,and their morphology was "hemispherical" or "saucer-like".The body was successfully separated.PKH26 stained exosomes and incubated with chondrocytes for 6 h.DAPI stained the nucleus,and marked exosomes were observed around the nucleus,marking the successful internalization of exosomes to chondrocytes.4.Screening the three sequences of YAP-si RNA,the inhibitory effect of si RNA-2 was the most obvious.The concentration of si RNA-2 was tested.It was found that the inhibitory effect was better at a concentration of 40 nmol / L.The detection time of si RNA-2 showed that After 48 h,the inhibitory effect can be maximized.Subsequent experiments used si RNA-2 at a concentration of 40 nmol / L for 48 h to silence YAP.5.Effects of different concentrations of exosomes on chondrocyte m RNA and protein expression.1)BMSCs-Exo were transfected into chondrocytes at concentrations of 0,1,5,10 ?g / m L.The results showed that COL-?,Aggrecan and SOX9 all showed an upward trend with increasing concentration.Among them,SOX9 increased significantly,and Aggrecan and COL-? increased significantly at higher concentrations(10 ?g / m L)(P <0.05 or P <0.01).According to WB,the expression levels of Aggrecan,COL-? and SOX9 increased in a concentration-dependent manner.Compared with 0 ?g / m L,SOX9 and Aggrecan were significantly increased(P <0.05 or P <0.01),and COL-? was significantly increased(P <0.01)except for low concentration(1 ?g / m L).2)R-BMSCs-Exo were transfected into chondrocytes at concentrations of 0,1,5,10 ?g / m L.The results showed that,except for SOX9,Aggrecan and COL-? showed an increasing trend with increasing concentration.Aggrecan and COL-? increased significantly(P <0.01);SOX9 increased significantly at higher concentrations(10 ?g / m L)(P <0.05).WB detection showed that Aggrecan,COL-? and SOX9 showed a concentration-dependent trend,and the expression levels increased in turn.COL-? increased significantly(P <0.01);except for low concentrations(1 ?g / m L)of Aggrecan and SOX9,protein expression increased significantly(P <0.05 or P <0.01).3)Comparing the RNA expression levels of BMSCs-Exo(E-10)and R-BMSCs-Exo(RE-10)at 10 ?g / m L,Aggrecan,COL-?,and SOX9 compared with 0 ?g / m L 10 The change is not obvious,and the change of RE-10 is significant(P <0.05 or P <0.01).Compared with SO-10,SOX9,RE-10 increased significantly(P <0.05).According to WB,the protein expression of Aggrecan,COL-? and SOX9 at 0,E-10 and RE-10 showed an increasing trend.Compared with 0 ?g / m L,E-10 and RE-10 were significantly increased in Aggrecan and COL-?(P <0.05 or P <0.01).SOX9 increased significantly in RE-10(P <0.01).6.Effects of different concentrations of exosomes on chondrocyte proliferation and migration.1)The CCK-8 method detects the proliferation of chondrocytes at 0,E-10 and RE-10.Compared with 0 ?g / m L,E-10 cells have a stronger proliferative capacity(P <0.05);RE-10 cells The proliferation is significant,and it is in a logarithmic growth period(P <0.01).Compared with E-10,RE-10 cells proliferated significantly(P <0.05).It can be seen from the foregoing that both E-10 and RE-10 can promote the proliferation of chondrocytes,which is better than R-BMSCs-Exo.2)Transwell method detects the migration of chondrocytes at 0,E-10 and RE-10.Compared with 0 ?g / m L,the migration ability of E-10 cells is significantly enhanced(P <0.05);Significantly improved(P <0.01).Compared with E-10,the migration ability of RE-10 cells was significantly improved(P <0.05).As can be seen from the foregoing,both E-10 and RE-10 can promote the migration of chondrocytes,which is better than R-BMSCs-Exo.7.RNA detection of YAP and its target genes Ankrd1 and CTGF found that RNA expression at 0,E-10 and RE-10 showed an increasing trend.Compared with 0 ?g / m L,the expression of E-10 increased in all three,but the difference was not statistically significant;RE-10 increased significantly(P <0.05).Compared with E-10,CTGF increased significantly(P <0.05).According to WB,compared with 0 ?g / m L,YAP,Ankrd1 and CTGF increased significantly at E-10 and RE-10(P <0.05 or P <0.01);compared to E-10,the expression level of RE-10 increased High,but the difference is not obvious(P> 0.05).As can be seen from the foregoing,both BMSCs-Exo and R-BMSCs-Exo can promote the expression of YAP and its target genes Ankrd1,CTGF,and R-BMSCs-Exo has a better effect.8.After silencing YAP with si RNA,his target genes Ankrd1 and CTGF were also significantly inhibited(P <0.05),and the expression levels of cell phenotype-related genes Aggrecan,COL-? and SOX9 were also significantly reduced(P <0.05 or P <0.01),Indicating that cell division and proliferation are significantly inhibited,and YAP directly promotes the expression of genes related to cell proliferation.When YAP was silenced and then intervened with exosomes,the expression and proliferation of YAP and its target genes CTGF,Ankrd1,phenotype-related genes Aggrecan,COL-? and SOX9 were also significantly reduced(P <0.05 or P < 0.01),which proves that exosomes promote Aggrecan,COL-? and SOX9 gene expression was inhibited by YAP.The WB results indicate the same problem.9.After silencing YAP with si RNA,the effect of exosomes on the proliferation and migration of cartilage femoral cells.1)The values of each group were detected by CCK-8 method.The results showed that YAP-si RNA significantly inhibited the proliferation of chondrocytes(P <0.05);when YAP was silenced and then intervened with exosomes,its proliferation effect was significantly reduced(P <0.01).Exosomes promote chondrocyte proliferation by regulating YAP.2)Transwell migration experiments confirmed that the migration of chondrocytes decreased significantly after YAP was silenced(P <0.05);when YAP was silenced and then intervened with exosomes,the migration effect was significantly lower than that of pure exosomes(P < 0.01).As can be seen from the foregoing,exosomes promote chondrocyte migration by up-regulating YAP.Conclusions:1.BMSCs-Exo and R-BMSCs-Exo can promote the proliferation,migration and phenotype maintenance of chondrocytes,and the effect of R-BMSCs-Exo is better.2.Exosomes promote chondrocyte proliferation,migration and phenotype maintenance by up-regulating YAP. |
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