Experimental study on the effect of osteoblast-derived exosomes stimulated by mycobacterium tuberculosis lysate on osteoclasts

English Keywords:Exosomes  Osteoblasts  Osteoclasts  Mycobacterium tuberculosis lysate

English Abstract:

  【Abstract】 Objectives: To study the effect of osteoblast-derived exosomes(MTL-OB-Exo) stimulated by mycobacterium tuberculosis lysate(MTL) on the formation of osteoclasts. Methods: Cell Counting Kit-8 (CCK-8) was used to determine that 35.9ng/ml was the optimal dose of MTL, which was used to stimulate mouse osteoblasts. Differential centrifugation was used to separate and extract MTL-OB-Exo and normal mouse osteoblast-derived exosomes(OB-Exo). The morphology and size were observed by transmission electron microscopy, and Western blot(WB) was used to detect the expression of exosomal transmembrane protein CD9 and osteoblast-specific alkaline phosphatase(AKP). Mouse mononuclear macrophages(RAW264.7), the precursor cells of osteoclasts, were divided into three groups according to different intervention methods: experimental group, MTL-OB-Exo (10ng/ml); control group, OB-Exo (10ng/ml); blank group, no treatment. After 4 days of culture, RAW264.7 was observed under light microscope and tartrate resistant acid phosphatase(TRAP) staining was used. The morphology of osteoclasts in each group was observed and counted, and the difference in the number of osteoclasts was compared. In the same intervention group, the cells after intervention were cultured on the bone grinding plate for 10 days. The morphology of bone resorption lacunae in each group was observed and counted by methylene blue staining, and the difference in the number of bone resorption lacunae was compared. Results: MMTL-OB-Exo was of a round or round-like structure with a diameter of 30-100nm, and it highly expressed CD9 and AKP proteins. After the intervention of RAW264.7 in different ways, the number of osteoclasts formed in the experimental group was 39.60±1.95, which was significantly higher than that in the control group(30.20±1.64) and the blank group(28.80±1.58), and the difference was statistically significant(P<0.05). The number of bone resorption lacunae in the experimental group was 21.40±1.52, which was significantly higher than that in the control group(16.20±1.30) and the blank group(14.40±1.52), and the difference was statistically significant(P<0.05). Conclusions: MTL-OB-Exo can induce and enhance the formation of osteoclasts, result in bone destruction.