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-  2017 

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

DOI: doi:10.7507/1002-1892.201705088

Keywords: 微小RNA-140, 壳聚糖, 软骨细胞, 信号肽,

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Abstract:

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

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