The Study On Chemokines Production In Murine Microglia BV₂ Cell Induced By Recombinant Borrelia Burgdorferi Membrane Protein A

Objective:1. To use the murine microglia BV2 cell line as cell model to explore the stimulating effects of recombinant Borrelia burgdorferi membrane protein A (rBmpA).2. To use the chemokine chip, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PT-PCR technology to study the production of chemokines from microglial cells stimulated by rBmpA.3. Finally, the study is to explore the mechanism of Lyme neuroborreliosis caused by Borrelia burgdorferi.Methods:1. BV2 cells were cultured in DMEM-high glucose medium supplemented with 5% calf serum and seeded in in 96-well plates (3×105/mL,100μL per well). After 6-24h incubation in the medium, the cells were stimulated with 10μg/mL and 20μg/mL rBmpA respectively. The cellular supernatants were collected after 6,12, and 24 hours stimulation, and cell lysates were prepared using the RNAiso Plus reagent.2. Detecting the chemokine chip was used to detect chemokines in cell cultural supernatants. The volume of each sample is 150μL.3. Chemokines (CXCL2, CCL22, CCL5, and CXCL13) concentrations in the cell cultural supernatants were detected by ELISA.4. Specific primers were designed to amplify the gene CXCL2, CCL22, CCL5, and CXCL13. Chemokines (CXCL2, CCL22, CCL5, and CXCL13) mRNA expressions were assessed by quantitative real-time PCR, with data normalized to the housekeeping gene GAPDH.Result:1. Compared with the normal control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant were increased significantly after the rBmpA stimulation. The concentration of chemokines increased with the rBmpA addition.2. Compared with the normal control group, the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells were increased significantly with 10μg/mL and 20μg/mL rBmpA stimulation respectively.3. Compared with the normal control group, the CXCL13 was not changed after the rBmpA stimulation.Conclusion:1. Chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA, secreted from the BV2 cells. Thus, the production of chemokines in Lyme neuroborreliosis may be mainly from microglias.2. The rBmpA up-regulated the secretion of chemokines, such as CXCL2, CCL22, and CCL5 from the BV2 cells. Therefore, the rBmpA may be closely related with the development of Lyme neuroborreliosis.