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目的 探讨核定位信号肽偶联核激酶底物短肽(nucleus localization signal linked nucleic kinase substrate short peptide,NNS)修饰壳聚糖(chitosan,CS) (NNSCS),介导人微小RNA-140 (microRNA-140,miR-140)基因转染,对体外培养的兔关节软骨细胞的作用.方法将重组质粒GV268-miR-140和空质粒GV268分别与NNSCS复合形成NNSCS/pDNA纳米复合物.取新生新西兰大耳白兔膝关节软骨,采用胰蛋白酶和胶原酶联合消化法分离培养原代软骨细胞.取第2代软骨细胞分为3组:正常细胞对照组(A组)、NNSCS/GV268空质粒转染组(B组)、NNSCS/GV268-miR-140转染组(C组),B、C组细胞分别以NNSCS/GV268及NNSCS/GV268-miR-140纳米复合物瞬时转染.转染后,实时荧光定量PCR (real-time fluorescent quantitative PCR,RT-qPCR)检测外源miR-140的表达;Annexin V-FITC/PI双染色法及MTT法分别检测外源miR-140对软骨细胞凋亡及增殖活力的影响;RT-qPCR检测软骨细胞中Sox9、聚集蛋白聚糖(Aggrecan)、组蛋白去乙酰化酶4(histone deacetylase4,Hdac4)基因表达.结果RT-qPCR检测示,C组外源miR-140表达水平较A、B组明显上调(P＜0.05).与A、B组比较,C组软骨细胞凋亡率明显降低,细胞增殖活力明显增加,细胞内Sox9、Aggrecan基因相对表达量明显上调、Hdac4基因相对表达量明显下调(P＜0.05);A、B组间以上指标比较,差异均无统计学意义(P＞0.05).结论NNSCS可携带外源基因进入软骨细胞并高效表达,高表达的miR-140能提高体外培养软骨细胞的生物活性,为其用于治疗软骨损伤性疾病提供了实验依据.“,”Objective To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro.Methods Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes,respectively.Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits.The second generation chondrocytes were divided into 3 intervention groups:normal cell control group (group A),NNSCS/GV268 empty plasmid transfection group (group B),and NNSCS/GV268-miR-140 transfection group (group C).NNSCS/GV268 and NNNSCS/GV268-miR-140 complexes were transiently transfected into cells of groups B and C.After transfection,real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140;Annexin V-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes;the expressions of Sox9,Aggrecan,and histone deacetylase 4 (Hdac4) were detected by RT-qPCR.Results RT-qPCR showed that the expression ofmiR-140 in group C was significantly higher than that in groups A and B (P＜0.05).Compared with groups A and B,the apoptosis rate in group C was decreased and the proliferation activity was improved,Sox9 and Aggrecan gene expressions were significantly up-regulated,and Hdac4 gene expression was significantly down-regulated (P＜0.05).There was no significant difference in above indexes between groups A and B (P＞0.05).Conclusion Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently,the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro,which provides important experimental basis for the treatment of cartilage damage diseases.