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目的 探讨白细胞介素I受体拮抗剂 (IL 1ra)基因治疗小鼠Ⅱ型胶原关节炎效果 ,为临床开展类风湿关节炎基因治疗奠定基础。方法 通过基因工程方法将人IL 1racDNA插入真核表达载体pcDI中 ,构建成在真核细胞内表达IL 1ra的质粒pcDI IL 1ra。经酶切、PCR和DNA测序鉴定均证实了插入片段的正确性。体外和体内转染真核细胞后 ,分别用ELISA和免疫组织化学方法检测到了IL 1ra的高效表达。将小鼠分成 4组 ,每组 8只 ,1组、2组用基因枪给药法 (2 0 μg 只小鼠 ) ,3组、4组用肌肉注射法 (2 0 0 μg 只小鼠 ) ,一次性将这一构建质粒注射于Ⅱ型胶原诱导的DBA 1小鼠关节炎模型肌肉组织中。结果 用两种方式IL 1ra基因给药后第 4天 ,小鼠肌肉组织中IL 1ra表达结果经计算机图像分析 ,吸光度 (A) 4 90值 :基因枪组为 0 5 2± 0 0 3,肌注组为 0 4 8± 0 0 2 ,对照组 0 4 1± 0 0 2。基因枪组与于对照组比较P <0 0 1,肌注组与对照组比较P <0 0 5。给药后第 6天和第 12天 ,小鼠血清IL 1ra水平 ,与对照组比较基因枪组于第 6天和第 12天均显著升高 (P <0 0 1)。肌注组小鼠血清IL 1ra水平在第 6天和第 12天也显著高于对照组 (P <0 0 1,P <0 0 5 )。两种治疗组的小鼠病情从第 6天开始缓解 ,关节红肿较对照组减轻。第
Objective To investigate the effect of interleukin-1 receptor antagonist (IL-1ra) gene therapy on type II collagen arthritis in mice and lay a foundation for the clinical treatment of rheumatoid arthritis gene therapy. Methods Human IL 1 cDNA was inserted into the eukaryotic expression vector pcDI by genetic engineering to construct plasmid pcDI IL 1ra that expressed IL-1ra in eukaryotic cells. The correctness of the inserted fragment was confirmed by enzyme digestion, PCR and DNA sequencing. Eukaryotic cells were transfected in vitro and in vivo and the high expression of IL-1ra was detected by ELISA and immunohistochemistry respectively. The mice were divided into 4 groups of 8 mice in each group. Group 1 and group 2 were administered with a gene gun (20 μg of mouse), 3 and 4 groups were intramuscularly injected (200 μg of mouse) This constructed plasmid was injected into the muscle tissue of type II collagen induced DBA 1 mouse arthritis model at one time. Results The expression of IL-1ra in mouse muscle tissue was analyzed by computer image on the 4th day after administration of IL-1ra gene in two ways. The absorbance (A) 490 value was 522 ± 0 0 3 in the gene gun group 0 48 ± 0 0 2 for the infusion group and 0 4 1 ± 0 0 2 for the control group. Gene gun group compared with the control group P <0 01, intramuscular injection group compared with the control group P <0 05. The levels of IL-1ra in serum of mice on the 6th day and the 12th day after administration were significantly higher than those of the control group on the 6th day and the 12th day (P <0.01). The level of IL-1ra in the intramuscular group was also significantly higher than that in the control group on the 6th day and the 12th day (P <0.01, P <0.05). The mice in both treatment groups began to relieve their symptoms on the 6th day. The joint swelling was relieved compared with the control group. No.