CAJ | 학술논문

目的：探讨脾源性酪氨酸激酶syk在单增李斯特菌（LM）感染小鼠腹腔巨噬细胞对炎症复合体形成过程中的作用。方法将小鼠腹腔巨噬细胞随机分为BAY处理组、SB处理组、WO处理组、未处理组以及阴性对照组（NI），每组设3个复孔。BAY处理组、SB处理组、WO处理组分别用syk抑制剂BAY117082、P38MAPK抑制剂SB203580以及PI3K抑制剂wotamine预处理小鼠腹腔巨噬细胞1 h，除阴性对照组外，各组巨噬细胞感染LM 24 h， ELISA检测上清液中白细胞介素（IL）-18表达；免疫荧光观察细胞内炎症复合体的关键蛋白组分ASC-speck的形成情况；Western blot检测LM感染小鼠腹腔巨噬细胞不同时相点syk的磷酸化水平。结果 BAY处理前后阴性对照组的小鼠腹腔巨噬细胞的IL-18表达水平无明显变化（P>0.05）。BAY处理组小鼠腹腔巨噬细胞感染LM后IL-18的表达水平与未处理组相比明显降低（P<0.05），而SB处理组和WO处理组、未处理组间小鼠腹腔巨噬细胞的IL-18表达水平差异无统计学意义（P>0.05），免疫荧光结果显示BAY处理组小鼠腹腔巨噬细胞内ASC-speck阳性的细胞百分比明显低于未处理组（P<0.01）。syk磷酸化水平在LM感染5、15、30 min时均明显升高。结论 syk介导的信号通路参与调控LM感染过程中炎症复合体的形成。
목적：탐토비원성락안산격매syk재단증리사특균（LM）감염소서복강거서세포대염증복합체형성과정중적작용。방법장소서복강거서세포수궤분위BAY처리조、SB처리조、WO처리조、미처리조이급음성대조조（NI），매조설3개복공。BAY처리조、SB처리조、WO처리조분별용syk억제제BAY117082、P38MAPK억제제SB203580이급PI3K억제제wotamine예처리소서복강거서세포1 h，제음성대조조외，각조거서세포감염LM 24 h， ELISA검측상청액중백세포개소（IL）-18표체；면역형광관찰세포내염증복합체적관건단백조분ASC-speck적형성정황；Western blot검측LM감염소서복강거서세포불동시상점syk적린산화수평。결과 BAY처리전후음성대조조적소서복강거서세포적IL-18표체수평무명현변화（P>0.05）。BAY처리조소서복강거서세포감염LM후IL-18적표체수평여미처리조상비명현강저（P<0.05），이SB처리조화WO처리조、미처리조간소서복강거서세포적IL-18표체수평차이무통계학의의（P>0.05），면역형광결과현시BAY처리조소서복강거서세포내ASC-speck양성적세포백분비명현저우미처리조（P<0.01）。syk린산화수평재LM감염5、15、30 min시균명현승고。결론 syk개도적신호통로삼여조공LM감염과정중염증복합체적형성。
Objective To clarify the role of syk kinase in inflammasome activation in mouse peritoneal macrophages during Listeria monocytogenes (LM) infection. Methods Murine peritoneal macrophages were randomly divided into BAY treatment group, SB treatment group, WO treatment group, no treatment group and negative control group (NI). There were three wells in each group. The syk inhibitor BAY 117082, P38MAPK inhibitor SB203580 and PI3K inhibitor wotamine were used to treat murine peritoneal macrophages for 1h in BAY treatment group, SB treatment group and WO treatment group. Murine peritoneal macrophages were infected with LM for 24 h except NI group. The protein level of interleukin (IL)-18 in the supernatant was detected by ELISA kit. The activation condition of key molecule ASC in the infected-macrophages cyto-plasm was observed under fluorescence microscope. The phosphorylation levels of syk protein kinase at different time points during LM infection were determined by Western blot assay. Results There was no significant difference in IL-18 protein level before and after BAY treatment in NI group (P>0.05). The IL-18 protein level was significantly lower after LM infec-tion in BAY treatment group compared with that in no treatment group (P<0.05). In contrast, there was no significant differ-ence in IL-18 production between SB treatment group, WO treatment and no treatment group (P>0.05). Meanwhile, the per-centage of ASC-speck positive cells was obviously diminished in BAY treatment group compared with that in no treatment group (P<0.01). The phosphorylation levels of syk were significantly increased in 5 min, 15 min and 30 min post-infection. Conclusion Syk kinase signaling is involved in the inflammasomes activation upon Listeria monocytogenes infection in mu-rine macrophages.