可磷酸化短肽偶联壳聚糖介导IL-1RA与IGF-1共转染对兔关节软骨细胞的作用

探讨可磷酸化短肽偶联壳聚糖(phosphorylatable short peptide coupled chitosan,pSP-CS),介导人白细胞介素-1受体拮抗剂基因(interleukin-1 receptor antagonist protein,IL-1RA)和人胰岛素样生长因子1基因(insulin-like growth factor-1,IGF-1)共转染,对体外培养的兔关节软骨细胞的作用.将pSP-CS与共表达质粒p Bud CE4.1-IL-1RA+IGF-1、单基因表达质粒p Bud CE4.1-IL-1RA、p Bud CE4.1-IGF-1和空质粒p Bud CE4.1制成pSP-CS/p DNA复合物,转染体外分离培养的正常兔原代关节软骨细胞.ELISA法检测IL-1RA和IGF-1的表达,以表征pSP-CS转染效率;Cell Counting Kit-8(CCK-8)法分析软骨细胞的增殖活力;流式细胞仪检测软骨细胞的凋亡;定量PCR检测软骨细胞中基质金属蛋白酶抑制剂-1(matrix metallo-proteinase inhibitor-1,Timp-1)、基质金属蛋白酶-3(matrix metalloproteinase-3,Mmp-3)、聚集蛋白聚糖(Aggrecan)基因表达.转基因组IL-1RA和IGF-1有较高的表达水平;各转基因组明显促进细胞增殖、抑制细胞凋亡、下调Mmp-3基因表达、上调Timp-1和Aggrecan基因表达,且双基因组作用明显优于单基因组(P0.05).结果表明,pSP-CS可以携带外源基因进入软骨细胞并大量表达,IGF-1与IL-1RA协同作用明显提高体外培养软骨细胞的生物活性,为今后研究pSP-CS介导多基因体内治疗软骨损伤提供了基础.

Effects of IL-1RA and IGF-1 Gene Co-transfection with Phosphorylatable Short Peptide-conjugated Chitosan on Rabbit Articular Chondrocytes

The effects of phosphorylatable short peptide conjugated chitosan (pSP-CS) for the transfection of interleukin-1 receptor antagonist protein (IL-1RA) combined with insulin-like growth factor-1 (IGF-1) in rabbit articular chondrocytes was investigated in vitro. Co-expression plasmid pBudCE4.1-IL-1RA+IGF-1, single gene expression plasmid pBudCE4.1-IL-1RA and pBudCE4.1-IGF-1 and empty plasmid pBudCE4.1 were compared. IL-1RA and IGF-1 in the medium were assayed by ELISA. The proliferation and apoptosis of transfected chondrocytes were analyzed by Cell Counting Kit-8 and flow cytometry. Levels of matrix metalloproteinase-3(Mmp-3), matrix metallo-proteinase inhibitor-1(Timp-1) and Aggrecan mRNA evaluation were detected by quantitative real-time RT-PCR (RT-qPCR). We found that IL-1RA and IGF-1 productions in the medium were higher in non-control groups, which enhanced proliferation, reduced apoptosis, up-regulated Timp-1 and Aggrecan expression, and down regulated Mmp-3 expression were detected. The changes in double gene transfection group were more significant over the control (P<0.05). The results showed that pSP-CS carried exogenous genes into chondrocytes nearly to the efficiency of lipofectamine 2000. IL-1RA combined IGF-1 improved the biological activity of chondrocytes in vitro.