| Objective:MicroRNA-140?miR-140?plays an important role in the differentiation and metabolism of chondrocytes.However,the effect and mechanism of miR-140 in the repair process of cartilage defects need to be further studied.Here,we investigated the effect and mechanism of nucleus localization signal linked nucleic kinase substrate short peptide-conjugated chitosan(NNSCS)mediated the miR-140gene transfer on articular cartilage defects in rabbits.Methods:MiR-140 was amplified from human genomic DNA isolated from whole blood and was inserted into GV268 vector to generate recombinant plasmid GV268-miR-140.The nucleus localization signal linked nucleic kinase substrate short peptide?NNS?was conjugated to chitosan to form NNSCS,then NNSCS was combined with pEGFP-C1 vector to form NNSCS/pEGFP complexes.Chondrocytes were isolated from articular cartilage of newborn rabbits and were identified by toluidine blue staining.NNSCS/pEGFP complexes were transiently transfected into the second generation chondrocytes.The transfer efficiency of NNSCS was measured by GFP positive cells.In vitro:recombinant plasmid GV268-miR-140 and negative control plasmid GV268 were respectively combined with NNSCS to form NNSCS/GV268-miR-140 and NNSCS/GV268 complexes.The second generation chondrocytes were inoculated in cell culture plates,and the experiment was divided into 3 groups:transgenic group?group A?,negative control group?group B?and normal cell control group?group C?.NNSCS/GV268-miR-140 and NNSCS/GV268 complexes were respectively transiently transfected into cells of group A and B.The same mass volume of NNSCS was added in group C.After transfection,real-time fluorescent quantitative PCR?RT-qPCR?was used to detect the expression of miR-140;MTT assay and Annexin V-FITC/PI double labeling assay were respectively used to detect the effects of miR-140 on proliferation and apoptosis of chondrocytes;RT-qPCR and western blot were respectively used to detect the expressions of chondrogenic related gene Sox9,Aggrecan and histone deacetylase 4?Hdac4?at mRNA and protein level.In vivo:eighteen healthy male New Zealand white rabbits were randomly divided into 3 groups:transgenic group?group A?,negative control group?group B?and sham-operated group?group C?,n=6.Full-thickness cartilage defects were made in the articular surface of the right lateral femoral trochlea of the knee in group A and B;the right lateral femoral trochlea articular surface was only exposed in group C.After 1 week of the surgery,intra-articular injection of NNSCS/GV268-miR-140 and NNSCS/GV268 complexes were respectively administrated 2 times a week for 7weeks in group A and group B,the same volume isotonic saline in group C.At 8weeks after the surgery,the rabbits were sacrificed and analyzed.The expressions of miR-140,Sox9,Aggrecan and Hdac4 were detected by RT-qPCR;the repair of cartilage in the defect zone was evaluated by HE staining,safranine O/fast green staining and Aggrecan immunohistochemistry.Results:The GV268-miR-140 eukaryotic expression plasmid was successfully constructed,and combined with NNSCS to form NNSCS/GV268-miR-140 complexes.NNSCS could carry pEGFP-C1 into the chondrocytes,since about 40%of the cells were GFP-positive.In vitro:MTT showed that the proliferation activity in group A?150.3±10.3%?was significantly improved compared with those in group B?107.3±5.3%?and C?100.0±5.6%??P<0.05?.Flow cytometry showed that the apoptosis rate in group A?7.05±0.263%?was significantly decreased compared with those in group B?10.37±0.163%?and C?9.87±0.093%??P<0.05?.RT-qPCR showed that the expression of miR-140 in group A?5.87±0.543?was significantly higher than those in group B?1.27±0.143?and C?1.00±0.110??P<0.05?;miR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan gene mRNA?Sox9:4.97±0.558,Aggrecan:3.00±0.140?and obviously down-regulated the expression of Hdac4 gene mRNA?0.63±0.060?compared with those in group B?Sox9:1.30±0.340,Aggrecan:1.23±0.080,Hdac4:1.08±0.063?and C?Sox9:1.00±0.130,Aggrecan:1.00±0.070,Hdac4:1.00±0.030??P<0.05?.The results of western blot were consistent with RT-qPCR.MiR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan protein?Sox9:0.592±0.010,Aggrecan:2.287±0.117?and obviously down-regulated the expression of Hdac4 protein?0.451±0.008?compared with those in group B?Sox9:0.460±0.012,Aggrecan:1.593±0.009,Hdac4:0.752±0.025?and C?Sox9:0.464±0.008,Aggrecan:1.507±0.057,Hdac4:0.754±0.007??P<0.05?.In vivo:RT-qPCR showed that the expression of miR-140 in group A?2.603±0.334?was significantly higher than those in group B?0.882±0.105?and C?1.022±0.225??P<0.05?;miR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan gene mRNA?Sox9:3.697±1.042,Aggrecan:2.726±0.430?compared with those in group B?Sox9:0.919±0.078,Aggrecan:0.880±0.104?and C?Sox9:1.008±0.143,Aggrecan:1.008±0.132??P<0.05?.The mRNA level of Hdac4 gene in group B?4.434±1.187?was highest,and the others were in group A?2.552±0.182?and C?1.055±0.362?;miR-140 overexpression in group A obviously down-regulated the expression of Hdac4 gene mRNA compared with that in group B?P<0.05?.Cartilage repair appeared in group A,showing positive staining of HE,safranine O/fast green and Aggrecan;fibrous tissue proliferation and inflammatory cell infiltration were seen in the defect zone in group B without significant cartilage repairing.Conclusions:?1?Exogenous gene could be carried into the chondrocytes by NNSCS and expressed efficiently.?2?MiR-140 could improve the biological activity of chondrocytes cultured in vitro.?3?Intra-articular injection of NNSCS/GV268-miR-140complexes could improve the repairing of articular cartilage defect,which may be related to the up-regulation of Sox9 and Aggrecan gene expressions and the down-regulation of Hdac4 gene expression. |
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