The Reseach On The Role Of Recombinant Borrelia Burgdorferi Membrane Protein A (rBmpA) In Stimulating Human Leukemia T Lymphocyte Cell Line In Vitro And Kunming Mice In Vivo Effects

Objective:1. Using the Human leukemia T lymphocyte Jurkat cell line as cell model to explore the stimulating effects of recombinant Borrelia burgdorferi membrane protein A (rBmpA), screeing for the chemokine which secretion level is higher, may have relationship with Lyme disease closely for further research.2. To use the ELISA to detect the secretion of the chemokines that screened from the Jurkat cell:IL-2、IL-4、IL-6、IL-17A、TNF-a in the Human leukemia T lymphocyte cell line Jurkat cell culture supernatant, which stimulated by recombinant Borrelia burgdorferi membrane protein A (rBmpA), to explore the mechanism of Lyme arthritis caused by rBmpA in the cellular level.3. Using Borrelia burgdorferi recombinant membrane protein A (recombinant Borrelia burgdorferi membrane protein A, rBmpA) stimulate joint modeling in mice, applying the Real Time PCR (RT-PCR) to detect the expression of the chemokines that screened from the experiment:(MIP-1 α/CCL3) and (SDF-1 α/CXCL12) of mice, to explore the mechanism of Lyme arthritis caused by rBmpA in the gene level.Methods:1. Jurkat cells were cultured in RPMI-1640 medium supplemented with 5%calf serum and seeded in 24-well plates (5×10s/mL,1ml per well). After 6-24h incubation in the medium, then cells adaptation. the cells were stimulated with 100μg/mL PHA and 80μg/mL rBmpA respectively. The cellular supernatants were collected after 12 and 24 hours stimulation.2. Cytokines (IL-2、IL-4、IL-6、IL-17A and TNF-a) concentrations in the cell cultural supernatants were detected by ELISA.3. Using Kunming mice divided into three groups:Experimental group(0.005mg/ml). Control group (0.01MNaCl) Black group(Do not deal), Left and right legs are 50ul, at 7 days and 14 days to collect synovial fluid of mice blood and eyes, and then detected the expression of (MIP-1 a /CCL3) and (SDF-1 α/CXCL12) in the organization by RT-PCR.4. Specific primers were designed to amplify the gene CCL3 and CXCL12. Chemokines (CCL3 and CXCL12) mRNA expressions were assessed by quantitative real-time PCR, with data normalized to the housekeeping gene GAPDHResults:1. The research successful exploration of in vitro cultured human leukemia T cell lines (jurkat cell) conditions, methods and precautions.2. Mice model is successful established a for Lyme arthritis by Borrelia burgdorferi membrane protein A (rBmpA).3. According to ELISA results:80μg/rBmpA and 100μg/mL of PHA mL can be induced leukemia T lymphocyte cell line (jurkat cell) activation of the release of cytokines secreted IL-4 and IL-17A and The difference was statistically significant.Therefore, the rBmpA may be closely related with the development of Lyme diease.4. ELISA result:rBmpA can induce the mice animal models of Lyme arthritis and joint homogenate to produce Content of IL-17. The results that:the experimental group (rBmpA group) produced content of IL-17 was significantly higher than the control (PBS group) and control group.5. Antibody microarray high-throughput results:rBmpA can stimulate the mouse macrophage cell a variety of chemokines, Successfully screened the secretion level of MIP-1 a/CCL3 and SDF-1 a/CXCL12 To study.6. The mice serum supernatant and joint homogenates mRNA of CCL3 real-time Q-PCR results show that:The mRNA expression of CCL3 gene are differences between the three groups, Experimental group (rBmpA group), control group (PBS group) and black group. The expression of rBmpA group were higher than those in the experimental group and the control group, the difference was statistically significant.7. The mice joint homogenates mRNA of CXCL16 real-time Q-PCR results show that: The mRNA expression of CCL3 gene are differences between the three groups, Experimental group (rBmpA group), control group (PBS group) and black group.Conclusion:1. The research successfully explored the method in vitro of human T lymphocyte cell line (jurkat cells), at the same time successfully established animal models of Lyme arthritis in mice.2. The effects of rBmpA protein in vivo and in vitro was demonstrated that:the results indicated the rBmpA may be closely related with the development of Lyme arthritis.