钙结合蛋白S100A8、 S100A9在大鼠肺泡巨噬细胞中的表达及作用

Expressions and effects of calcium-binding protein S100A8 and S100A9 in alveolar macrophages of rats

目的观察钙结合蛋白S100A8、S100A9在大鼠肺泡巨噬细胞(AM)中的表达,及其对大鼠AM炎症介质释放的影响。方法分离、培养正常大鼠AM,分别给予不同剂量的S100A8及S100A9处理大鼠AM6h和12h,采用免疫组织化学和原位杂交方法观察大鼠AM中S100A8/A9蛋白和S100A8mRNA、S100A9mRNA的表达情况;采用酶联免疫吸附法检测大鼠AM上清液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和IL-8的浓度。结果(1)大鼠AM的细胞质与细胞膜均可见S100A8mRNA、S100A9mRNA和S100A8/A9蛋白表达。(2)给予不同剂量S100A8、S100A9处理大鼠AM,随着S100A8、S100A9剂量逐渐增加,大鼠AM上清液中TNF-α、IL-6和IL-8的浓度亦逐渐增加,各组之间比较差异均有统计学意义(P值均<0.05)。(3)给予相同剂量S100A8、S100A9处理大鼠AM不同时间,12h组大鼠AM上清液中TNF-α、IL-6和IL-8的浓度较6h组均明显增高(P值均<0.05)。(4)给予相同剂量S100A8和S100A9处理大鼠AM相同时间,S100A8组大鼠AM上清液中TNF-α和IL-8的浓度均高于S100A9组(P值均<0.05);而较高剂量的S100A8组细胞上清液中IL-6的浓度也高于相同剂量的S100A9组(P<0.05)。结论S100A8、S100A9可在大鼠AM中表达。S100A8、S100A9呈剂量、时间依赖性刺激大鼠AM分泌TNF-α、IL-6和IL-8等炎症介质。S100A8刺激大鼠AM分泌TNF-α、IL-6和IL-8等炎症介质作用明显强于S100A9。

Objective To observe the expressions of calcium binding proteins S100A8 and S100A9 in alveolar macrophages ( AM) of rats and their effects on the release of inflammatory mediators in AM of rats. Methods AM were isolated from rats and cultured in -vitro . Different doses of S100A8 and S100A9 were treated to AM for six hours or 12 hours. Immunohistochemistry and in situ hybridization were performed to observe the expressions of S100A8/A9 protein, S100 A8 mRNA and S100A9 mRNA in AM of rats. Enzyme linked immunosorbent assay was performed to detect the concentrations of tumor necrosis factor α( TNF-α), interleukin-6 (IL-6 ) and IL-8 in supernatant of rat AM. Results (1 )S100A8 A9 protein, S100A8 mRNA and S100A9 mRNA were expressed in the cytoplasm and on the cell membrane of rat AM.(2)Different closes of S100A8 and S100A9 were used to treat AM. The concentrations of TNF-α, IL-6 and IL-8 in supernatant of AM were correspondingly increasing with the increasing doses of S100A8 and S100A9. The differences between the groups were statistically significant (all P<0.05).(3)The same dose of S100A8 and S100A9 was ad mi nistered to treat the rat AM for different times. The concentrati ons of TNF-α, IL-6 and IL-8 in supernatant of AM in the 12-hour group were significantly higher than those in the 6- hour group (all P<0. 05).(4 ) AM were treated with the same dose of S100A8 and S100A9 for the same time. The concentrations of TNF α and IL-8 in the supernahirn of AM in the S100A8 group were higher than those in the S100A9 group ( all P<0. 05). The conce nlration of IL-6 in the supernatant of AM in the higher dose of S100A8 group was also higher than lhat in the same dose of S100A9 group ( P <0.05). Conclusions S100A8 and S100A9 can be expressed in AM of rats. S100 A8 and S100A9 stimulate the secretion of inflammatory mediators: TNF α, IL 6 and IL-8 in the AM of rats in time-dependence and conceniration-dependenee. The slimulani effect of S100A8 on the secretion of TNF-a. IL-6 and IL-8 in AM of rats is significanlly stronger than that of S100A9.