肺炎链球菌热休克蛋白40通过p38MAPK和JNK通路诱导小鼠巨噬细胞免疫应答  

Pneumococcal HSP40 induces the immune response in mouse macrophages via p38MAPK and JNK signaling pathways

作　　者：吴盈盈[1] 高松[1] 马峰[1] 崔晶晶[1] 姚华[1] 孙潇雨[1] 王继超[1] 胥文春[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《细胞与分子免疫学杂志》2015年第6期730-735,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31270984);重庆市自然科学基金(cstc2012jj A10009)

摘　　要：目的探讨肺炎链球菌热休克蛋白40(HSP40)引起小鼠巨噬细胞免疫应答的机制。方法表达、纯化重组HSP40蛋白(r HSP40),去除其中的脂多糖(LPS)。r HSP40处理C57BL/6野生小鼠骨髓来源巨噬细胞(BMDM)后,反转录PCR检测BMDM中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β、IL-23p19、IL-12p40、IL-12p35、IL-10的mRNA水平,ELISA检测TNF-α、IL-6、IL-12p40水平;r HSP40刺激后,ELISA检测野生型、Toll样受体2(TLR2)和TLR4缺陷的BMDM中TNF-α和IL-6的表达水平;丝裂原活化蛋白激酶(MAPK)抑制剂预处理BMDM后,ELISA检测其对r HSP40诱导TNF-α和IL-6水平的影响;Western blot法检测p38MAPK和c-Jun氨基末端激酶(JNK)磷酸化水平。结果获得纯度90%以上的r HSP40;r HSP40可显著增强p38MAPK、JNK的磷酸化水平和TNF-α、IL-6的表达;p38MAPK和JNK抑制剂可显著抑制TNF-α和IL-6的表达;与野生BMDM相比较,TLR4缺陷的BMDM表达TNF-α和IL-6显著降低。结论 HSP40诱导小鼠巨噬细胞产生免疫应答受JNK及p38MAPK信号通路调控,并且此过程依赖TLR4。Objective To investigate the mechanism of immune response in mouse macrophage induced by Pneumococcal heat shock protein 40 （HSP40）. Methods After recombinant HSP40 （rHSP40） underwent expression detection and purification, lipopolysaccharide （LPS） was removed from it. Then rHSP40 was used to stimulate bone marrow derived macrophages （BMDMs） derived from C57BL/6 wild-type mice. The mRNA levels of tumor necrosis factor α （TNF-α）, interleukin 6 （ IL-6）, IL-1β, IL-23p19, IL-12p40, IL-12p35 and IL-10 in BMDMs were determined by reverse transcription PCR; the expressions of TNF-α, IL-6 and IL-12p40 were measured by ELISA. After stimulated by rHSP40, the levels of TNF-α and IL-6 expressed by wide-type, TLR2-/- and TLR4-/- BMDMs were detected by ELISA. The effects of the pretreatment of mitogen-activated protein kinases （MAPK） inhibitors on the secretion of TNF-α and IL-6 induced by rHSP40 were also evaluated by ELISA in BMDMs. The phosphorylation levels of p38MAPK and c-Jun N-terminal kinase （JNK） were determined by Western blotting. Results The rHSP40 protein reached a purity of more than 90%. It significantly enhanced the phosphorylation leve4s of p38MAPK and JNK as well as the expressions of TNF-α and IL-6. The p38MAPK and JNK inhibitors significantly suppressed the expressions of TNF-α and IL-6. The expressions of TNF-α and IL-6 in TLR4-/- BMDMs significantly decreased compared with wide-type BMDMs. Conclusion HSP40-induced immune response of mouse macrophages is regulated by p38MAPK and JNK signaling pathways, and this induction process depends on TLR4.

关 键 词：肺炎链球菌 巨噬细胞 热休克蛋白40 p38分裂原活化蛋白激酶 C-JUN氨基末端激酶 

分 类 号：R378.14[医药卫生—病原生物学] R392.12[医药卫生—基础医学]