Infection Of Macrophages With Viable Strain H37Ra And Heat-killed H37Rv

Objective：To study the difference in the expression of immunesubstances after inoculating mice with viable M.tuberculosis H37Ra andHeat-Killed M. tuberculosis H37Rv， and to compare the differencebetween viable and Heat-Killed M.tuberculosis in the induction ofimmune responses and related signaling pathways, and thus to learnwhether viable or Heat-Killed bacteria could be a possible candidate fornew vaccines.Methods:1. Peritoneal macrophages from BALB/c mouse werecultured with H37Ra and H37Rv for10hours respectively, to observe thesituation of each group macrophages, and calculate the phagocytic rates ofmacrophages by the acid dye staining method.2. BALB/c mice wereabdominally immunized with viable H37Ra, Heat-Killed H37Rv and NS,respectively. At30days after immunization, the macrophages wereisolated. The expression levels of IL-12p40,TNF-a, IFN-γ weredetermined by RT-PCR and ELISA，The expression levels of NO、H₂O₂ were determined by Griess and Chemical method;3. BALB/c mice wereabdominally immunized with viable H37Ra, Heat-Killed H37Rv and NS,respectively. At30days after immunization, the macrophages wereisolated. and the changes of CD40L induced IFN-γ were detected by flowcytometry, laser scanning confocal microscope and Western bolt.Results：1.The phagocytic rates of macrophages were（55.71±8.42）%for viable M.tuberculosis and （14.82±2.12）%for Heat-KilledM.tuberculosis with a significant difference（P<0.01）.2.The mRNA levelsof1L-12p40，TNF-a，IFN-γwere higher in Viable M. tuberculosis H37Rathan those of in Heat-Killed M.tuberculosis, the cytokine levels ofIL-12p40，TNF-a，IFN-γ, NO and H₂O₂were higher in Viable M.tuberculosis H37Ra than those of in Heat-Killed M.tuberculosis,3. Theexpression of CD40L on macrophages induced by IFN-γ in viableM.tuberculosis stimulated group was higher than that of Heat-KilledM.tuberculosis stimulated group.Conclusion：Viable M.tuberculosis H37Ra can induce the activation ofmacrophages and the production of more protective immune substances,which play important roles in the host immune response, so it would be acandidate for tuberculosis vaccine. At the same time, IFN–γ canstimulate the viable M.tuberculosis CD40L signal channel generates animmune response, However, Heat-Killed M. tuberculosis H37Rv can’tinduce the activation of macrophages effectively and produce less protective immune substances, which makes it unlikely to be a candidatefor tuberculosis vaccine...