莱姆关节炎昆明小鼠动物模型的建立

Successful creation of an animal model of Lyme arthritis in KM mice

目的使用伯氏疏螺旋体菌悬液对昆明小鼠进行胫跗关节皮下注射,建立昆明小鼠莱姆关节炎动物模型。方法将40只昆明小鼠随机分为PBS组、BSK组、实验组Ⅰ和实验组Ⅱ,每组10只,分别经双后肢胫跗关节皮下注射相应菌悬液或対照液,每周观察小鼠健康变化,测量小鼠双后肢胫跗关节左右径,并进行足及胫跗关节X光检查,记录相关数据。饲养6周后,将小鼠脱颈处死,取双后肢胫跗关节进行组织病理学检查及IL-1β和TNF-ɑ相关检测,对两种方法建立的昆明小鼠莱姆关节炎模型进行评价。培养基中培养,每种抗生素分别设立20ug/ml(高浓度),2ug/ml(中浓度)和0.2ug/ml(低浓度)三种浓度。提取细菌总RNA,采用Real-time PCR分析MRSA ccrA、ccrB、ccrC基因的表达水平。结果 PBS组及BSK组小鼠在实验期间未出现关节红肿现象,小鼠活动自如。实验组Ⅰ和实验组Ⅱ注射1周后小鼠胫跗关节出现肿胀,肿胀持续1周后逐渐减轻,但实验组Ⅱ小鼠减轻程度低于实验组Ⅰ小鼠。实验组Ⅰ和实验组Ⅱ小鼠注射后6周时胫跗关节组织研磨液中TNF-ɑ和IL-1β水平升高,分别为(44.07±12.07)pg/ml、(18.59±4.514)pg/ml和(47.17±8.387)pg/ml、(20.37±5.381)pg/ml。病理学检查实验组Ⅰ和实验组Ⅱ小鼠关节间隙变窄,关节滑膜纤维组织增生,细胞层数增多,大量炎性细胞浸润,滑膜组织增厚。影像学检查实验组Ⅰ和实验组Ⅱ小鼠胫跗关节周围软组织肿胀,关节间隙毛糙变窄,呈高密度影,骨质破坏严重。结论 1×105/ml PBS伯氏疏螺旋体悬液和1×105/ml BSK伯氏疏螺旋体悬液均能诱导昆明小鼠莱姆关节炎的发生,以1×105/ml BSK伯氏疏螺旋体悬液诱发的关节炎更严重,更持久。

Objective To induce Lyme arthritis and create a model of Lyme arthritis in KM mice by hypodermically injecting a suspension of Lyme disease spirochetes（Borrelia burgdorferi）into the tibiotarsal joint of KM mice. Methods Forty mice were randomly divided into a PBS group,a BSK group,experimental group I,and experimental group II.Each group consisted of 10 mice.The PBS group was injected with PBS alone,the BSK group was injected with BSK medium alone,experimental group I was injected with 1×105 spirochetes per ml of PBS,and experimental group II was injected with 1×105 spirochetes per ml of BSK medium.Fiftyμl of the control solution or spirochete suspension was injected in both hind tibiotarsal joints of mice in each groups.After injection,dynamic changes were observed weekly.The size of the hind tibiotarsal joints was measured,X-rays of the foot and tibiotarsal joint were taken,and related data were recorded.Six weeks later,mice were sacrificed and the hind tibiotarsal joints were collected.Joints were examined histopathologically and an enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of TNF-ɑand IL-1βin the synovial fluid of the tibiotarsal joint.Two methods of creating a model of Lyme arthritis in KM mice were assessed.Results Mice in the PBS group and BSK group had no redness or swelling and they behaved normally during the period of this study.One week after injection,mice in experimental groupⅠand experimental groupⅡhad obvious redness and swelling of the tibiotarsal joint that gradually subsided after 7days.These manifestations were more evident in groupⅡ.The level of TNF-ɑin the PBS group was 12.90±1.342pg/ml,that in the BSK group was 14.33±3.424pg/ml,that in experimental group I was 44.07±12.07pg/ml,and that in experimental group II was 47.17±8.387pg/ml.The level of IL-1βin the PBS group was 11.70±2.239pg/ml,that in the BSK group was 10.78±2.284pg/ml,that in experimental group I was 18.59±4.514pg/ml,and that in experimental group II was 20.37±5.381pg/ml.Levels of TNF-ɑand IL-1βin the synovial fluid of the tibiotarsal joint both increased significantly in experimental groupsⅠandⅡ.Specifically,levels of TNF-ɑand IL-1βin experimental groupⅡwere higher than those in experimental groupⅠ.Histopathology revealed narrowing of the joint space,hyperplasia of fibrous tissue and an increase in layers of cells in the synovial membrane,infiltration of large numbers of inflammatory cells,and thickened tissue of the synovial membrane in experimental groupⅠand experimental groupⅡ.X-rays revealed opacities where swelling of soft tissue around the tibiotarsal joint,roughness of the articular surface,and narrowing of the joint space were evident.Severe bone destruction was evident.