腺病毒介导的内皮抑素对关节炎大鼠炎症及细胞因子的影响

The effect of endostatin mediated by adenovirus on the inflammation and cytokines of arthritis rats

目的研究重组腺病毒介导的人内皮抑素(nAD-GFP-ES)对大鼠Ⅱ型胶原诱导型关节炎(CIA)炎症及细胞因子的影响。方法首先,扩增并纯化重组腺病毒。然后,建立CIA模型,自造模次日治疗组给予rAD-GFP-ES(1×10^(11)pfu·kg^(-1)·周^(-1)×4周),各组大鼠每周进行一次关节炎指数(AI)评分,于造模第4周取血清行Western Blot检测大鼠体内内皮抑素(ES)、白细胞介素(IL)-1D、肿瘤坏死因子(TNF)-α的表达并计算相对含量。结果①纯化后的rAD-GFP-ES和rAD-GFP滴度分别为：6．6×10^(12) pfu/m1．4．8×10^(12)pfu/ml；吸光度(A)_(260nm)/A_(2180nm)均＞1．3。②感染rAD-GFP-ES的大鼠体内ES获得了稳定表达,是非模型组的2．4倍。③感染rAD-GFP-ES可以降低大鼠AI评分及IL-1β、TNF-α的表达。结论①rAD-GFP- ES注射可使ES基因在大鼠体内获得稳定表达。②感染rAD-GFP-ES能明显降低模型大鼠的AI评分。③IL-1β、TNF-α共同促进CIA大鼠炎症的形成和发展,rAD-GFP-ES可能通过抑制IL-1β、TNF-α的表达而发挥抗炎的作用。

Objective To investigate the effect of recombinant adenovirus mediatied human endostatin （rAD-GFP-ES） on rats with collagen type Ⅱ induced arthritis （CIA）, and explore the mechanism of inflamma- tion and cytokines inhibition on rats CIA. Methods The rAD-GFP-ES was amplified and purified. The model of rat CIA was induced by intradermal injection of type Ⅱ collagen combined with complete Freund＇s adjuvant （CFA）. On the second day after the injection, the therapeutic administration of rAD-GFP-ES （1×10^11 pfu·kg ^-1·week^-1×4 weeks） were performed to the rats. The mean arthritis index （AI） was scored every, week since then. The relative concentrations of ES, IL-1β, TNF-α in sera collected at the fourth week were evaluated by western blotting. Results ①The titer of the purified rAD-GFP-ES and rAD-GFP was 6.6×10^12 pfu/ml and 4.8×10^12 pfu/ml, respectively （A260nm/A280nm〉1.3）. ②The concentration of ES in sera of the group treated with rAD-GFP-ES was 2.4-fold higher compared to the normal group.③The mean arthritis index of the group treated with rAD-GFP- ES was much lower than that of the model group. The administration of rAD-GFP-ES could significantly deereas the production of IL-1β and TNF-α in sera. Conclusions①The rAD-GFP-ES is efficiently expressed in vivo. ②The rAD-GFP-ES has an inhibitory effect on the arthritis index of rat CIA. ③IL-1β and TNF-α are involved in the pathogenesis of RA. The rAD-GFP-ES has an inhibitory effect on the expression of IL- 1β and TNF-α in rat CIA.