可磷酸化短肽偶联壳聚糖介导IGF-1和IL-1RA双基因联合治疗兔关节软骨损伤

非病毒基因转移载体--壳聚糖被广泛用于基因转染，然而相对较低的转染效率限制了其在基因治疗中的应用.本课题组曾经报告可磷酸化短肽修饰壳聚糖(hosphorylatable short peptide coupled chitosan，pSP-CS)可增加体外培养细胞的DNA转染效率.本研究中采用pSP-CS作为基因载体介导人白细胞介素-1受体拮抗剂基因(interleukin-1 receptor antagonist gene, IL-1RA)和人胰岛素样生长因子-1基因(insulin-like growth factor 1 gene, IGF-1) 局部转染, 联合治疗兔关节软骨损伤.将pSP-CS与单基因表达质粒pBudCE4.1-IGF-1、pBudCE4.1-IL-1RA和共表达质粒pBudCE4.1-IGF-1+IL-1RA制成pSP-CS/pDNA复合物，制备股骨外侧髁全层软骨损伤模型，pSP-CS/pDNA 复合物关节腔内注射4周. ELISA分析发现，转基因组关节腔灌洗液中含有大量外源蛋白IGF-1和IL-1RA. 定量PCR检测mRNA显示, 各转基因组明显下调基质金属蛋白酶-3(matrix metallo-proteinase-3, Mmp-3)基因表达; 上调基质金属蛋白酶抑制剂-1(matrix metallo-proteinase inhibitor-1, Timp-1)和二型胶原(Collagen II) 基因表达(P < 0.05)；双基因转染组作用明显优于单基因转染组(P< 0.05). HE及Collagen II免疫组化染色显示, 各转基因组软骨损伤处出现不同程度的软骨性修复，以双基因转染组作用最优. 本研究表明，pSP-CS可以携带外源基因进入软骨组织并局部大量表达，IGF-1与IL-1RA协同作用明显促进损伤软骨修复，为今后临床多基因治疗软骨损伤提供了实验基础.

Phosphorylatable Short Peptide-Conjugated Chitosan-Mediates IGF-1 and IL-1RA Double Gene Transfection to Repair Injured Articular Cartilage in Rabbits

Chitosan as a non-viral gene transfer vector has been widely used for gene transfection, however the relatively low transfection efficiency limits its further use in gene therapy. We have previously reported that phosphorylatable short peptide coupled-chitosan (pSP-CS) can improve the transfection efficiency of the DNA-chitosan (DNA/CS) complex in cultured cells. In this study, we investigated the effects of pSP-CS mediated human interleukin-1 receptor antagonist gene (IL-1RA) and human insulin-like growth factor 1 gene (IGF-1) local transfection on the repair of injured articular cartilage in rabbit. Single gene expression plasmid pBudCE4.1-IGF-1, pBudCE4.1-IL-1RA, and co-expression plasmid pBudCE4.1-IGF-1+IL-1RA were combined with pSP-CS to form pSP-CS/pDNA complex. Full-thickness cartilage defects were made on the articular surface of the lateral femoral condyle, then pSP-CS/pDNA complexes were injected into the joint cavity for 4 weeks. ELISA analysis revealed that the synovial fluid of knee joint in transgenic group contains large amounts of exogenous proteins of IGF-1 and IL-1RA. Quantitative PCR analysis showed that the transgenic complexes resulted in up-regulated the expression of Timp-1 and Collagen II and down-regulated the expression of Mmp-3 (P <0.05), the roles of the co-expression transfection group were superior to those of single-gene transfection group (P <0.05). Hematoxylin and eosin (HE) histochemistry and Collagen II immunohistochemistry staining revealed that pDNA/pSP-CS treatment resulted in varying degrees of hyaline-like cartilage repair in each transgenic group, the co-expression transfection group produced greater effects than the single-gene transfection group. The results show that pSP-CS can effectively carry exogenous genes into cartilage tissue and highly expressed locally. The combination of IL-1RA and IGF-1 can significantly improve the repair of articular cartilage injury in vivo, which provides a basis for future clinical research multi-genes therapy of articular cartilage injury.