白介素I受体拮抗剂基因治疗小鼠Ⅱ型胶原关节炎的疗效观察  

Effect of interleukin-I receptor antigonist gene therapy on arthritis induced by type II collagen in mice

作　　者：杨高云[1] 狄春辉[2] 蒋明[1] 李芳[2] 张颖妹[2] 袁岚[2] 宋泉声[2] 马大龙[2] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院风湿免疫内科,100730 [2]北京医科大学卫生部医学免疫学重点实验室

出　　处：《中华医学杂志》2002年第14期990-994,共5页National Medical Journal of China

摘　　要：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。Objective To investigate the effect of interleukin 1 receptor antagonist gene therapy on type II collagen induced arthritis in DBA/1 mice. Methods Plasmid pcDI IL 1ra that expresses IL 1ra in eukaryotes was constructed by inserting human IL 1ra cDNA into the eukaryotic expression vector pcDI. Eukaryotes were transfected with the plasmid pcDI IL 1ra in vivo and in votro. The expression of 8 IL 1ra was examined by ELISA and immunohistochemistry. Type II collagen was used to induced arthritis in 32 DBA/1 mice. This plasmid was injected into the muscles of DBA/1 mice with arthritis induced by type II collagen by gene gun (20 μg/mouse) and into the muscle of 8 mice by intramuscular injection (200 μg/mouse). After the administration, the condition of arthritis was observed. The serum IL ira was examined 6 and 12 days after administration. The expression of IL ira in muscles was tested by computerized imaging analysis. Results PCR and DNA sequencing proved the accuracy of the inserted fragment. ELISA and immunohistochemistry detected high expression of IL ira in vivo and in vitro. The absorbance ( A ) 490 value of IL ira in the mouse musle was 0 52±0 03 in gene gun group, and 0 48±0 02 in intramuscular injection group, all higher than that in the control group (0 41±0 02, P <0 01 and P <0 05). The serum IL ira values in the gene gun group and intramuscular injection group 6 days and 12 days after therapy were all significantly higher than that in the control group (all P <0 01; and P <0 01 and P <0 05). Since the 6 th day after therapy, the redness and swelling of joints in both therapies groups were alleviated. Pathological examination made 12 days after therapy showed relief at different degrees of the infiltration of imflammatory cells, hyperplasia of synovia, bone infiltration, and cartilage destruction, especially in the gene gun group. Conclusion Gene therapy of IL ira via non virus eukaryotic expression vactor, especially by gene gun, is effective in treating

关 键 词：白细胞介素受体 基因疗法 小鼠 类风湿关节炎 

分 类 号：R593.22[医药卫生—内科学]