Establishment Of Osteoclast Model Infected By Brucella In Virulence Evaluation

Brucellosis is a zoonotic disease caused by Brucella.It is category B infectious disease and class II animal epidemic disease.Animals and people could show symptoms such as bone and joint disease so call brucellosis osteoarthritis.Brucellosis arthritis could occur in acute and chronic stages of Brucellosis,and the clinical manifestations in the acute stage shows pain,joint swelling,in chronic stage severe deformation of bone and joint tissue could be seen.Researchs have confirmed that osteoclast infiltration and cartilage destruction appear in the swollen tail joint of Brucella infected gene deletion mice,suggesting that osteoclasts play an important role in the pathogenesis of brucellosis arthritis.In this study,the osteoclast induction method and the model of Brucella infected osteoclasts were established in vitro,and the interaction effect between different virulence Brucella strains with osteoclasts was evaluated by this model,in order to elaborate the pathogenesis of brucellosis arthritis,and the risk of Brucella induced arthritis was measured by the activation of osteoclast function as reference to evaluate the virulence of Brucella,results and conclusions are as follows:First,in order to obtain stable osteoclast cell and eliminate the disturbling factors such as cell source,RAW264.7 cells were used as the object for osteoclasts inducing.Osteoclast cell lines were induced by using RANKL and M-CSF and the preparation and use of culture medium is established.The cells after inducement shows positive of tartrate resistant phosphatase stain,the generation of bone resorbing pits,and the osteoclast function related genes TRAP,MMP-9,Cathepsin K and RANK were significantly up-regulated（P<0.05）,which were 10.7 times,12.4 times,10.8 times and 16 times respectively.As a result,the osteoclast model induced in vitro was established.Second,in order to establish the model of osteoclast infect by Brucella,Brucella vaccine strain A19 which can cause calf arthritis was selected to establish the infection model.Firstly,the osteoclasts were infected with Brucella A19-GFP under different infection MOI,and the replicate kinetics of Brucella in osteoclasts was evaluated by the change of unit fluorescence area;Secondly,the average maturity and function of osteoclasts were measured by the average TRAP staining area and bone resorbing area of osteoclasts.The results shows that the ability of osteoclasts to resorb bone slices was significantly improved（P<0.05）,all the bone resorbing area over 30000μm~2;Finally,the transcription levels of TRAP,Cathepsin K,MMP-9,RANK,IL-6 and IL-8 were detected.The transcription of MMP-9 and TRAP were significantly up-regulated by 7 and 3 times which shows Brucella infection affects the function of osteoclasts.Third,in order to evaluate the risk of bone tissue damage caused by different virulence Brucella strains,this study infected osteoclasts by selecting Brucella S2,M5,A19 and 2308 strains,and evaluated the affect of different strains of Brucella infection on osteoclast function.The results shows that the area of osteoclasts infected by Brucella 2308 strain was 55301μm~2.The average coloring area is 989μm~2.The transcription of MMP-9 increased 4.85 times,which was significantly higher than vaccine strains（S2 and A19）.The strong correlation between MMP-9 response and Brucella species differentiation factors was confirmed by Two-way ANOVA of variance of gene transcription level caused by Brucella infection and RAW 264.7 cell induction（P<0.01）.It shows the reliability of this model to measure the risk of Brucella induced bone injury.As a result,a stable Osteoclast cell modle was established by the induce of RAW264.7 cells and the risk of bone injury caused by different strains of Brucella was analyzed.It shows that Brucella 2308strain could significantly activate the osteoclast function,and the vaccine strain Brucella has effect on the osteoclast function which lays a foundation for measuring the bone injury and elaborate the pathogenic mechanism of Brucella arthritis,in order to eliminate the side effects of new Brucella vaccine development.