蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制探讨

目的：通过体外实验探讨蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制。方法：提取蛆虫分泌物,用糜蛋白酶抑制剂VR23、丝氨酸蛋白酶抑制剂（PMSF）预处理,将未处理的蛆虫分泌物、与抑制剂预处理后的蛆虫分泌物、PBS分别加入小鼠前成骨细胞细胞系MC 3T3-E1进行培养,24、48、72 h后,以蛋白免疫印迹法检测纤连蛋白含量,以CCK-8法检测细胞活性。与金黄色葡萄球菌共培养后,通过细菌涂板计数及免疫荧光法检测胞内菌数量。实验数据采用SPSS 25.0软件包进行统计学分析。结果：蛆虫分泌物可以降解纤连蛋白,不对细胞活性产生影响。细菌涂板计数可见,经蛆虫分泌物处理的小鼠前成骨细胞细胞系中,胞内菌含量较经糜蛋白酶抑制剂或丝氨酸蛋白酶抑制剂预处理的蛆虫分泌物处理的小鼠前成骨细胞细胞系中胞内菌含量低,而经PBS处理的小鼠前成骨细胞细胞系中胞内菌含量最高。免疫荧光法检测亦得到相似结果。结论：蛆虫分泌物可用于减少金黄色葡萄球菌对小鼠前成骨细胞细胞系的内化作用,为慢性难治性骨髓炎的治疗提供了新的思路。

Mechanism of maggot excretion/secretion in anti-intracellular Staphylococcus aureus internalization of osteoblast: an in vitro study

PURPOSE: This study was aimed to explore the mechanism of excretion/secretion (E/S) in protecting osteoblast being internalized by Staphylococcus aureus through in vitro experiments. METHODS: Excretion/secretion(E/S), E/S pre-treated with serine protease inhibitors(PMSF), E/S pre-treated with chymotrypsin inhibitors VR23 and PBS were respectively added to the mouse pre-osteoblast cell line (MC 3T3-E1) for culture. After 24, 48, 72 hours, Western blot was used to detect the fibronectin degradation, and CCK-8 was used to detect cell viability. After 24 hours, the cell line was co-cultured with Staphylococcus aureus to form intracellular bacteria to observe whether intracellular bacteria had formed in different groups. Flat colony counting method and immunofluorescence method were used to observe intracellular bacteria. The data were analyzed by SPSS 25.0 software package. RESULTS: E/S degraded fibronectin in extracellular matrix without influencing cell viability. Through flat colony counting method, the least intracellular bacteria were observed in the cell line treated with E/S. In cell line treated with E/S pre-treated with PMSF or the chymotrypsin protease inhibitor, more intracellular bacteria were observed. In PBS treated cell line, the most intracellular bacteria were observed. These results showed statistically significant(P<0.05) and similar results were obtained from immunofluorescence photography. CONCLUSIONS: E/S can be used to reduce the effect of Staphylococcus aureus on the internalization of MC 3T3-E1, which provides a new idea for the treatment of chronic refractory osteomyelitis.