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.
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 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: 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。.
Keywords: MicroRNA-140; chitosan; chondrocytes; rabbit; signal peptide.