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本研究旨在测定金黄色葡萄球菌(简称金葡萄)L型能否刺激小鼠腹腔巨噬细胞产生α-肿瘤坏死因子(TNF-α)。将从临床标本中分离出的金葡菌肉汤培养物接种于5%NaCl琼脂平板上并涂布均匀,以氯苯唑青霉素纸片(每片1000μg)法诱导出典型的金葡菌L型,再移种于含氯苯唑青霉素500μg/ml的高渗肉汤培养基中。经小鼠腹腔注射灭活的化脓性链球菌A3,3天后收获腹膜渗出的巨噬细胞。以不同浓度的灭活金葡菌及其L型与巨噬细胞(2×10~5)共同孵育4或18h,测定培养上清液对L细胞的细胞毒活性。采用渗透溶解法分离L型细胞膜,并以酚-水混合物抽提胞膜作巨噬细胞免疫印迹分析,测定L型胞膜诱导产生TNF-α的活性。同时,将鼠巨噬细胞与L型
The purpose of this study was to determine if L-form of Staphylococcus aureus could stimulate the production of α-TNF in mouse peritoneal macrophages. Staphylococcus aureus broth cultures isolated from clinical specimens were inoculated on 5% NaCl agar plates and spread evenly. A typical S. aureus L-form was induced by a method of cloprofen penicillin (1000 μg per piece) , Then transplanted in hypertonic broth culture medium containing 500μg / ml of oxacillin. Peritoneal exudate macrophages were harvested three and three days after mice were injected intraperitoneally with S. pyogenes A3. Different concentrations of inactivated Staphylococcus aureus and its L-type and macrophages (2 × 10 ~ 5) were incubated with 4 or 18h, the cytotoxic activity of the culture supernatant on L cells was measured. The L-type cell membrane was separated by osmotic dissolution and the membrane was extracted by phenol-water mixture for macrophage immunoblot analysis to determine the activity of L-type membrane-induced TNF-α production. Meanwhile, murine macrophages and L-type