基于尿酸盐和三磷酸腺苷协同刺激痛风性关节炎发病新机制的动物模型构建和评估

Gout is an arthritis caused by hyperuriceamia, which is associated with IL-1β secretion by urate crystals (MSU) stimulation. Based on this theory, acute gouty arthritis models are mainly established by injecting MSU in segmental joint to cause local inflammatory, however, these models are non-specific inflammation, and not related to the level of serum uric acid. The characteristics and pathological manifestations of arthritis models are different from gout in human body. Previously, we discover that hominine gout result from synergistic reaction between MSU and adenosine triphosphate (ATP), and related to the genotype of ATP receptor（P2X7R）. Animal test showed that when injected ATP with MSU together, rapid development of multiple severe and painful joints swelling were caused, compared to local joint slight swelling only injected by MSU in rats. It indicated that the real gouty arthritis model is induced by co-stimulation of MSU and ATP. To establish a stable and repeatable model of gout, this program intends to screen out experimental rats with gout susceptibility genes by comparing the SNP genotype of P2X7R gene. Moreover, a stable hyperuricemia model was established by knocking out the gene of uricolase or inhibiting the uricolase with drugs. Eventually, we explore appropriate administration and dosage of MSU, ATP to induct experimental gouty arthritis models which similar to hominine gout, and screen reliable model by evaluating characteristics of arthritis, pathology, level of serum uric acid and degree of inflammation.

痛风是与高尿酸血症相关的关节炎，其发病与尿酸盐（MSU）刺激IL-1β分泌有关；基于此，目前主要采用关节注射MSU等方法建立痛风模型，但模型的关节炎为非特异性炎症，发病与血尿酸水平关系不大，关节炎特点、病理表现均与人体痛风存在差异。项目组前期发现，痛风发病需要MSU与ATP协同作用，是否发病与ATP受体（P2X7R）遗传背景有关；并且动物试验发现，与大鼠关节中注射MSU仅引起关节局部肿胀不同，同时注射ATP可出现与痛风类似的关节剧烈红肿，提示MSU和ATP协同诱导的关节炎才是真正痛风发作。为成功建立痛风模型，本项目拟通过比较P2X7R不同SNP基因型的功能差异，筛选出携带痛风易感基因型的大鼠；再通过敲除尿酸酶基因或药物抑制尿酸酶等方法，建立稳定的高尿酸血症模型；最后摸索适当的MSU、ATP给药途径和剂量诱发类似人体痛风的关节炎，通过评估关节炎特点、病理、血尿酸及炎症水平筛选可靠的痛风模型。

痛风是与高尿酸血症相关的关节炎，其发病与尿酸盐（MSU）刺激IL-1β分泌有关；基于此，目前主要采用关节部位注射MSU建立痛风模型，但模型关节炎发病与血尿酸水平关系不大，关节炎特点与人类痛风存在差异。项目组前期发现痛风发病需要MSU与ATP协同作用，故拟采用MSU和ATP共同构建痛风模型。由于痛风发病受遗传背景、环境等多方面影响，为构建稳定发病大鼠模型，需要摸索MSU、ATP给药途径和剂量，在携带痛风易感基因的高尿酸血症大鼠中构建痛风模型。项目主要研究内容有：筛选大鼠痛风易感基因、建立高尿酸血症模型、构建和评估痛风动物模型。项目取得重要结果有：（1）采用腹腔注射尿酸酶抑制剂和尿酸酶基因敲除两种方法建立了大鼠高尿酸血症模型，（2）在尿酸酶抑制或敲除的高尿酸血症大鼠中成功构建了痛风模型，（3）发现大鼠P2X7R基因1016位点等位基因A及基因型AA与大鼠痛风相关。由于高尿酸血症鼠血尿酸水平受喂养条件影响大，项目组摸索出在尿酸酶基因敲除鼠或药物抑制鼠中从尾静脉注射MSU和ATP的方法诱发痛风发病。具体为第一天从大鼠尾静脉注射MSU悬液（0.1g/mL），100ul/只；第二天再次尾静脉注射同等剂量MSU悬液，同时尾静脉注射ATP溶液（10mmol/L），200ul/只。通过这种方法可以使30%大鼠出现痛风发病，在携带P2X7R基因1016位点AA基因型大鼠中，73%可出现痛风发病；在携带1016位点AA基因型出现痛风发病的子代大鼠中，基本都可以诱导痛风发病。本项目构建模型关节炎特点与人类痛风类似：是建立在高尿酸血症基础上，在关节部位自发出现红肿疼痛，炎症在12小时达高峰，1周内自行缓解，病理主要为中性粒细胞浸润。本项目基于ATP和MSU协同作用构建大鼠痛风模型的成功，为验证ATP是痛风第二致病信号的假说提供了直接证据，为痛风新靶点药物开发和深入研究痛风发病机制提供了理论基础和实验平台。

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