The Effect Of Receptor Tyrosine Kinase Inhibitor On Repair Tissue In Chondrocyte And Osteoarthritis Cartilage

PartⅠIn-vitro research of the effect of receptor tyrosine kinase inhibitor genistein on the expression of cytokine interlukin-1β,NO and phenotype expression of collagen and proteoglycan of chondrocyteObjective: the obtained rabbit chondrocytes were cultured in vitro, after the cell were identified, then we use receptor tyrosine kinase inhibitor genistein as an interferential measure, after interference, the cytokine interlukin-1β,NO were tested. phenotype expression of collagenⅠ,Ⅱand proteoglycan were detected in three different phase(4 weeks,8 weeks,12 weeks), in this study we will explore the protective role of the receptor tyrosine kinase inhibitor in chondrocyte and osteoarthritis cartilage.Methods: articular chondrocytes were isolated from the four weeks-aged New Zealand rabbits and were cultured in vitro, the primary and subculture of chondrocytes were indentified by morphological changes and toluidine blue staining .then we selected fitful cells as the subject, the cultured cells were divided into 7 groups: control, LPS and LPS, different concentrations genistein (5μg/ml,10μg/ml,15μg/ml,20μg/ml and 25μg/ml). and at day 1,2,3 and 4, IL-1βlevel was tested by enzyme-linked immunosorbentassay (ABC-ELISA), NO level was detected by Griess method, using reverse transcription-polymerase chain reaction(RT-PCR), immunocytochemistry (ICC) and toluidine blue staining to understand the changes of phenotype expression of collagenⅠ,Ⅱand proteoglycan in chondrocyte in vitro.Results: primary cultured chondrocytes were observed under the inverted microscope, chondrocytes maintained the morphology and immunochemical staining pattern within three passages, and the ability of excretion and proliferation of chondrocytes were decreased, the passage cycle was prolonged. Low level expression of interlukin-1βwere observed in the control group, while increased expression of interlukin-1βwere observed in LPS group and LPS, different concentrations genistein groups, and there were no difference between those groups. In the control group there were no expression of nitric oxide(NO) were tested. Increasing level expression of NO were detected in the group stimulated with LPS. the increasing trend was decreased after the intervention of genistein, the downregulated level of NO was related to the concentration.Toluidine blue staining showed that chondrocytes in control group were positively stained in Cambridge blue and nucleolus in deep blue. Large aggregate of cells and extra cellular matrix were stained to purplish red, and nucleolus stained to deep blue. Toluidine blue staining of chondrocytes stimulated with LPS were Cambridge blue, in which some nucleolus were negatively stained, and staining of ECM were tint. The staining of other chondrocytes with intervention of genistein especially in groups of 20μg/ml and 25μg/ml concentrations was close to those of normal chondrocytes.results of RT-PCR and ICC showed that there were no significant expression of collagenⅠ, and with the stimulation of LPS, upregulating level expression of collagenⅠcould be detected, In groups of LPS, genistein of different concentrations, the expression trend of were similar to the LPS group, but the expression level were lower than group of LPS, compared in those groups, 20μg/ml and 25μg/ml were much decreased (P＜0.05). increased expression of collagenⅡcould be tested in all groups. compared with upregulated level of collagenⅡin different phase, the groups of control,LPS and LPS, genistein of 20μg/ml and 25μg/ml concentrations showed significant difference (P＜0.05)Conclusions: the rabbit chondrocytes cultured in vitro maintained the specific chondrocytes phenotypes in the first 3 passages and have enough activities and was suitable for this study. This part showed that genistein can effectively inhibit the expression of NO above certain concentration, but could not decreased the expression of IL-1βat any concentration, it can improve the expression of collagenⅡand increase the synthesis of proteoglycan. the expression level of collagenⅠwere under control. genistein helped to keep the specific chondrocytes.PartⅡAnimal model of OA and the effect of genistein on the expression of matrix metalloproteinase(MMPs)-1,13,IL-1βand NO in vivoObjective: Build animal model of osteoarthritis, and use this animal model to understand the role of metalloproteinase(MMPs)-1,13,IL-1βand NO in vivo, and the effect of genistein on the expression of MMP-1,13,IL-1βand NO. Methods: according to Hulth method, we built OA model and did X-ray exam and micro-anatomy observation at 4,8,12 weeks. Expression levels of MMP-1,13mRNA,IL-1βand NO at different phase were detected in both control and treatment groups.Results: X-ray exam and micro-anatomy observation showed the articular surface was smooth on the whole, the medial gap was slightly narrow with subchondrol sclerosis and enhanced bone density, but no spur formation at week 4. At week 8, typical sign of OA was seen, and late OA signs at t week 12. at 4 weeks, the expression of MMP-1,13mRNA showed a low level in all groups, and there were no significant difference(P＞0.05); at 8 weeks, the expression of MMP-1,13mRNA increased in all groups, but the upregulated levels in genistein groups (0.3mg/kg,0.4mg/kg and 0.5mg/kg) were less than the control group(P＜0.05); at 12 weeks, the expression of MMP-1,13mRNA in all groups were less than that at 8 weeks, higher than that at 4 weeks, compared with control groups, the unregulated level in genistein groups (0.3mg/kg,0.4mg/kg and 0.5mg/kg) have significant difference.Increasing expression of interlukin-1βwere observed in each groups, but there were no difference between those groups at the same phase. In the control group there were no expression of nitric oxide(NO) were tested. Increasing expression of NO were also detected in other group. but the increasing trend was decreased after the intervention of genistein(0.3mg/kg,0.4mg/kg and 0.5mg/kg), P＜0.05.Conclusions: X-ray exam and micro-anatomy observation showed that OA model at 4,8,12 weeks can present three different pathological phase(early,moderate,end-stage). overexpression of MMP-1,13 broken the balance of synthesis and degradation of ECM. Interference of genistein((0.3mg/kg,0.4mg/kg and 0.5mg/kg) can decrease the overexpression of MMP-1,13 at moderate,end-stage osteoarthritis. As same as that in vitro, the results showed that genistein can reduce the expression of NO in certain concentration, while did nothing to the expression of interlukin-1β.PartⅢIn-vivo research of the effect of genistein on the phenotype, histology and ultra-microstructure of osteoarthritis cartilage and chondrocyteObjective: to explore the effect of receptor tyrosine kinase inhibitor genistein genistein on the phenotype, histology and ultra-microstructure of osteoarthritis cartilage and chondrocyte.Methods: using New Zealand rabbits to build OA model, these were divided into 6 groups(control and 0.1mg/kg,0.2mg/kg,0.3mg/kg,0.4mg/kg,0.5mg/kg genistein) at 4,8 and 12 weeks after operation, cartilage sections were made and stained with HE/SOFG for Mankin score; the expression level of collagenⅠandⅡin matrix was tested by immunohistochemistry(IHC); the change of ultra-microstructure of cartilage was observed through TEM.Results: the Results of Mankin score showed significant difference between control group and intervention group, P＜0.01, at week 4 the experiment group had low expression of typeⅠcollagen, at week 8 the positive staining area of collagenⅠincreased, and the expression level could be much higher at week 12; the expression level of collagenⅠincreased more in the experiment group; the expression level of typeⅡcollagen decreased less in the experiment group than the control group; TEM showed that in experiment group at week 4 indicated normal or slight impairment; at week 8, indicated slight to medium injury; at week 12, appeared to be medium or severe injury. at each phase the control group appeared to be worse than the experiment group.Conclusion: the results of Mankin score and IHC showed that genistein partly increased the synthesis of collagenⅡ, and delayed the abnormal expression of collagenⅠ, the observation of ultra-microstructure of cartilage explored the alleviation of the ultra-microstructure damage of cartilage after injury.