摘要
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(phosphorylatable short peptide coupled chitosan,pSP-CS)可增加体外培养细胞的DNA转染效率.本研究采用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p Bud CE4.1-IGF-1、p Bud CE4.1-IL-1RA和共表达质粒p Bud CE4.1-IGF-1+IL-1RA制成pSP-CS/p DNA复合物,制备股骨外侧髁全层软骨损伤模型,pSP-CS/p DNA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 metalloproteinase-3,Mmp-3)基因表达;上调基质金属蛋白酶抑制剂-1(matrix metalloproteinase 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.
Chitosan has been widely used for DNA transfection as a non-viral gene transfer reagent. We previously showed that phosphorylatable short peptide coupled-chitosan（pSP-CS） improved the transfection efficiency in cultured cells. In this study,we usedpSP-CS to mediate human interleukin-1receptor antagonist gene（ IL-1RA） and human insulin-like growth factor 1 gene（ IGF-1） in local transfection in rabbit for repairing injured articular cartilage. Expression plasmid of p Bud CE4. 1-IGF-1and p Bud CE4. 1-IL-1RA,or double expression plasmid p Bud CE4. 1-IGF-1 ＋ IL-1RA were mixed withpSP-CS to formpSP-CS / pDNA complex. Cartilage damage was made on the articular surface of the lateral femoral condyle prior to then pSP-CS / pDNA complex was injected into the joint cavity. After 4 weeks,ELISA revealed that the synovial fluid of knee joints in the transgenic group contained high levels of exogenous proteins of IGF-1 and IL-1RA. Quantitative PCR showed up-regulation of Timp-1 and CollagenⅡ with down-regulation Mmp-3（ P〈0. 05） in the transgenic samples. The double-expression vector performed better（ P〈0. 05）. HE staining and Collagen Ⅱ immunohisto- chemistry indicated that hyaline-like cartilage repair occurred following pDNA /pSP-CS treatments. The results showed thatpSP-CS effectively transferred exogenous genes into cartilage tissues and co-expression of IL-1RA and IGF-1improved the repair of articular cartilage injury.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2015年第2期175-181,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
山东省自然科学基金资助项目(No.ZR2012HQ034)
国家自然科学基金资助项目(No.81301737)~~
