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

发布时间：2018-06-16 17:48

  本文选题：微小RNA- + 壳聚糖　； 参考：《中国修复重建外科杂志》2017年10期

【摘要】：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(P0.05)。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(P0.05);A、B组间以上指标比较,差异均无统计学意义(P0.05)。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。
[Abstract]:Objective to investigate the effect of nuclear localization signal peptide coupled nuclear kinase substrates localization signal linked nucleic kinase substrate short peptiden (NNSN) on rabbit articular chondrocytes cultured in vitro, and to mediate the transfection of human minimal RNA-140 micro RNA-140 miR-140 gene into chitosan chondrocytes. Methods the recombinant plasmids GV268-miR-140 and empty plasmids GV268 were combined with NNSCS to form the NNSN CSP / pDNA nanocomplexes. The articular cartilage of newborn New Zealand rabbits was isolated and cultured by trypsin and collagenase digestion. The second passage of chondrocytes were divided into three groups: normal control group (group A) was transfected with NNSCSP / GV268 empty plasmid, group B was transfected with NNSCSP / GV268-miR-140, group C was transfected with NNSCSP / GV268-miR-140 and group C was transiently transfected with NNSCSP / GV268-miR-140 nanocomplex, respectively. After transfection, Real-time fluorescent quantitative PCR RT-qPCR The expression of exogenous mi R-140 was detected by Annexin 鈪，

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