An in vitro study of the effect of Mycobacterium tuberculosis infection on the expression of bone destruction-related factors in osteoclasts

English Keywords:Mycobacterium tuberculosis  Osteoclasts  C-src  Matrix metalloproteinase-9(MMP-9)  Cathepsin K(CK)  Carbonic anhydrase 2(CA2)  Integrin-β3

English Abstract:

  【Abstract】 Objectives: To establish a model of Mycobacterium tuberculosis infection of osteoclasts(OC) and explore the mechanism of Mycobacterium tuberculosis infection on OC. Methods: Peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) were isolated from healthy volunteers. Receptor activator of nuclear factor-κB ligand(RANKL) and macrophage-colony stimulating factor(M-CSF) were used to make PBMCS into OC, and tartrate resistant acid phosphatase(TRAP) staining was performed on the cells. The constructed kanamycin resistant H37Rv pMV261-GFP green fluorescent strain was resuscitated and cultured with 10% oleic albumin dextrose catalase(OADC), 7H9 and kanamycin containing Mycobacterium tuberculosis special liquid medium in an incubator at 37℃ until the optical density(OD) value was about 0.5 at 600nm. The OC cells cultured alone were set as the blank control group. And OC cells were also infected with Mycobacterium tuberculosis at different multiplicity of infection(MOI) for 24h, and MTT colorimetric method was used to detect cell survival rate. The MOI with the highest cell survival rate was selected as experimental MOI, and OC cells infected with H37Rv at experimental MOI were set as the experimental group. Fluorescence microscopy and Mycobacterium tuberculosis acid-fast staining were used to observe the transfection of Mycobacterium tuberculosis at the experimental MOI. Quantitative real-time PCR(qRT-PCR) was used to detect the expressions of non-receptor tyrosine kinase C-src, cathepsin K(CK), carbonic anhydrase 2(CA2), Integrin-β3 and matrix metalloproteinase-9(MMP-9). Immunohistochemistry was used to detect the expressions of P-src, CK, CA2, Integrin-β3 and MMP-9 on the cell surface. Western blot(WB) was used to detect the protein expression levels of P-src, CK, CA2, Integrin-β3, and MMP-9. Results: TRAP staining showed that more than 90% of the cells were OC after 15d of culture, which could be used for experiments. The results of MTT colorimetric assay showed that the cell survival rate was the highest when the MOI was 20∶1(P<0.05). This transfection multiplicity can be used as the concentration of experimental group. Fluorescence microscopy showed that when the transfection multiplicity ratio was 20∶1, the green fluorescent Mycobacterium tuberculosis entered the OC and was successfully transfected into the OC. The results of acid-fast staining after infection of OC with Mycobacterium tuberculosis showed that when the MOI was 20∶1, the acid-fast Mycobacterium tuberculosis stained red entered OC and was also successfully transfected into OC. The results of qRT-PCR, cell immunohistochemistry, and WB showed that the expressions of MMP-9, CK, C-src, CA2, and Integrin-β3 in the experimental group were higher than those in the blank control group (P<0.05). Conclusions: Mycobacterium tuberculosis can transfect OC; Compared with the blank control group, the levels of five bone destruction factors in the experimental group transfected with OC by Mycobacterium tuberculosis were increased, suggesting that bone destruction of spinal tuberculosis may be related to this, which may provide a new exploration direction for the diagnosis and treatment of bone tuberculosis diseases.