Study Of The Chimera Models With Human Rheumatoid Arthritis Synovial Fibroblasts-cartilage-SCID Mice For Rheumatoid Arthritis

Background and objectiveRheumatoid arthritis (RA) is an autoimmune disease that predominantly targets the synovial joints and ultimately leads to joint destruction. The main clinical manifestation is symmetry, chronic, progressive polyarthritis, and can involve multiple organs. Its hallmark is that the synovial joints appear chronic inflammation、 hyperplasia, the formation of pannus and the articular cartilage、bone、ligament、 tendon appears progressive destruction, ultimately leads to joints deformity and loss of function. The incidence of RA in China is about0.4%,0.5%to1.0%globally [1], mainly in the young, the disease is high level of disability; seriously affect the work and the quality of life of patients. As for many other chronic disease, the cause and detail pathogenesis of RA remain a mystery. The RA animal models are the one of the best way to study the pathogenesis of RA.There are two types of traditional RA animal models:one is inducisibility arthritis models include adjuvant arthritis、collagen-induced arthritis, and so on. Anther one is spontaneous arthritis models include K/BXN mice, NZB/NZW mice [2]. Study found that all of above can simulate multiple arthritis, joint local swelling, severely can cause joint deformity. Pathological change can show synovial hyperplasia、articular cartilage and bone destruction inflammatory cell invasion. Although these characteristics show similar process with human RA at clinical manifestation, pathology and immunity, but the inflammatory reaction is only limited in the area of joints, with a certain self-limiting. There are hardly test the RF、the CCP antibody、inflammatory factory、MMPs in the blood serum. The spontaneous arthritis models can test above all, but the mice are expensive. RA is complex disease, these models can only reflect the "T cell-mediated chronic systemic inflammation response", not show the critical effect of synovial fibroblast in synovial hyperplasic、 chronic inflammation、joint destruction. These animal models with a certain self-limiting, were builded under specific conditions, can only focus on one or several factors, not fully reflect the characteristics of RA.With in-depth study of RA synovial fibroblasts (RASFs), it is found that RASFs are not only a "bystander", but also an "active participant" in the pathogenesis of RA[3]. In the RA, RASF have been always in a state of chronic activation, the cells appear a series of transformation features. The growth regulation of RASFs were obstacled, leading to cells appear "tumor-like growth" and excessive proliferation. The cells beside of the "T cell-depend way" can secrete a variety of cytokine, causing chronic persistent arthritis, synovial hyperplasic and articular cartilage and bone destruction directly. RASFs play a key role in the pathogenesis of RA.In the1990s, Geiler[4] first builded a novel humanized animal model. He implanted the RA synovial membrane and normal cartilage co-implantation into a severe combined immunodeficiency (SCID) mouse, discovered that the synovial membrane appear hyperplasy, the cartilage invasion and destruction. In the surface of cartilage can test mRNA of MMPs.From then, the method of models and the graft material have been improvement continuously. The synovial membrane、synovial fibroblasts、normal cartilage, even joint fluid can be choosed as the graft material; The transplantation can be the synovial membrane and cartilage co-implant, synovial fibroblasts and cartilage co-implant, can also be only the synovial fibroblasts; Transplant site was also diverse, included the kidney capsule、intraperitoneal、 subcutaneous and knee of the mouse[5～7]. The SCID—HuRAg animal models can be completely free from the influence of immune cells and inflammatory environment, showing the almost identical process in the pathomechanism of synovial membrane and cartilage with RA, and can be detected the CCP antibody、IL-6、TNF-α and other cytokines in serum.These new models can confirm that the crucial role of RASFs, improve the pathogenesis of RA, has an irreplaceable role in the pharmacological studies、new drug development, become the mainstream of RA animal models.In the domestic, the study of SCID—HuRAg models is later than that in abroad. There only several cases of report in SCID—HuRAg models.xiao-chang hong or zhu-ping [8、9] used synovial membrane and normal cartilage as a co-implantion into the ear or subcutaneous of SCID mouse, observed synovial hyperplasy and cartilage degradation. So we want to study of the chimer models with human RASF-cartilage-SCID mice for RA to observe the role of RASFs and how can they work in the SCID mouse. If the models study successful, we will fill the gaps and can use this novel model to screen the effect Chinese herbs.Methods(一)Setting up the systerm of RASF/OASF cultivation and identification1、The synovial fibroblasts primary culture was used the method of two enzymes (type Ⅱ collagenase and0.25%trypsin) combine digestion. The RA or OA synovial membrane was isolated、digested、cultured and passaged at37℃in5%CO2to obtain and build the system of the RASF/OASF cultivation and conservation.2、The synovial fibroblasts were used the methods of nature purification and relapse adherention to purify the cells. Then the cells were used the vimentin antibody and CD68antibody to identification.3、The purifical cells were used the5-bromodeoxyuridine(5-Brdu) to label. The concentration of10mmol/15-Brdu jointed into the cell culture bottle, then added10ml complete medium, the ultimate concentration of5-Brdu adjusted10umol/l, in5%CO2cell culture. The labeled synovial fibroblasts were used5-Brdu antibody to identificition.(二)The method of transplantation1、Chose the transplant cellsWe chose the forth passaged、labeled RASF/OASF as transplant cells. The cells were digested、centifugated and suspensed, at last the density of cells adjusted2×105/1.2、The human normal cartilageThe normal cartilages from one case of diabete gangrene amputate operation. We rejected the ankle joint cartilage, then clipped into a bulk of0.5cm×0.5cm.The cartilages transplanted into SCID mouse in3hours.3、Inert sterile gel spongeThe inert sterile gel sponges were clipped into the volume of0.8cm×0.8cm.4、Setting up the transplantation animal medols24SCID mice into two groups in random,12in each group. RASFs were injected into a cavity of inert sterile gel sponge, and then with the normal human cartilage co-implanted in the back subcutaneously of SCID mice. OASFs were control group.(三)Evaluation the transplantation animal models1、The graft paraffin section and H&E were made to evaluate the histological scores of grafts on cartilage invasion by synoviocytes and perichondrocytic cartilage degradation.2、The knee joint paraffin section and H&E were made to evaluate the synovial hyperplasia and cartilage invasion in knee joints of mice.3、We used the IHC to detect the expression of Brdu and Vimentin in synoviocytes.4、We used the ELISA to detect the serum level of human Interleukin-6(IL-6) and matrix metalloproteinase-3(MMP-3). Results(一)The results of synovial fibroblasts in the culture、identify and label.We obtained numbers of synoviocytes succefully.Then we used the methods of nature purification and relapse adherention, obtained numbers of purify synoviocytes. We idenfied and labeled the third passaged synoviocytes. At fluorescence microscope, the synovial fibroblasts to shows green, displayed vimentin positive, CD68negative.The cells morphological diversity, mainly in spindle-shape, the cellular located in the cell central, the nucleolus clear. The vitality of cells was good. In the microscope, the5-Brdu labeled cells show brown and yellow in the celluae. The rate of cells label was well.(二)Evaluated the transplantation animal models1、The evaluation of the graftsIn the microscope to observed the graft paraffin section:The synovial fibroblasts survivalled and proliferated around the cartilage. The cells invaded the cartilage, the invasion located at the edge of cartilage, the surface of cartilage rarely see the invasions. The chondrocyte smediated the cartilage degradation were clearer at the edge of cartilage, the morphological of chondrocytes have changed. In the RASF group, the histological scores of grafts on cartilage invasion by synoviocytes (0.643±0.690vs0.333±0.500), cartilage degradation (2.286±0.7661vs1.667±1.000) were higher than that of OASF group.2、The evaluation of the knee jointsIn the microscope to observed the knee joints paraffin section:In the mouse knee joints, the synovial hyperplasy and with new blood vessels information. Some new blood vessels and proliferated synovial can informed fibrous nodules. The hyperplasia synovial can invade the cartilage; even can invade the bone severely. In the RASF group, the sorces of synovial hyperplasia(3.091±0.837vs1.667±0.985p<0.01), cartilage invasion (1.636±1.748vs0.583±1.379p<0.05) were higher than that of OASF group. 3、The migration of synovial fibroblast in the SCID mouseIn the SCID mice subcutaneous found lots of Brdu and Vimentin markers positive synoviocytes, in the cartilage invasion of grafts can be deteceted manipulus positive synoviocytes, and in the knee joints bone marrow and hyperplasia of the synovial tissue can be detected single positive synoviocytes.4、The content of human IL-6and MMP-3in the serumThe serum level of IL-6was detected only one case in the RASFs group, its content was55pg/ml, OASF group was detected noting. The serum content of MMP-3in the RASF group was one case59pg/ml, OASF group (39.50+17.35)pg/ml.ConclusionIn summary, in the SCID mice can be successfully established RA animal models with RASFs and cartilage, confirmed the RASFs on cartilage grafts with invasion and degradation in vivo, cytokine can be secreated by the SFs,such as IL-6and MMP-3. RASFs through the blood to migrate to the synovial joints, and can induce inflammation in joints.Compared with the OASFs, RASFs with stronger capacity in cell proliferation、invade and destructived the cartilage.The OASFs can secrete more content of MMP-3than RASFs in the blood serum. We also successfully set up the cultural and consecrate system of RASF/OASF.