核定位信号肽偶联核激酶底物短肽修饰壳聚糖介导微小RNA-140对兔关节软骨细胞作用的研究

Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes

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)(^(NNS)CS),介导人微小RNA-140(micro RNA-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^(NNS)CS复合形成^(NNS)CS/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)、^(NNS)CS/GV268空质粒转染组(B组)、^(NNS)CS/GV268-miR-140转染组(C组),B、C组细胞分别以^(NNS)CS/GV268及^(NNS)CS/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mi R-140的表达;AnnexinⅤ-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 deacetylase 4,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 ^(NNS)CS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 NNSCS/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 of miR-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.