中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
11期
983-988
,共6页
周洲%杨科%陈丽丽%陈虹亮%李忠玉%吴移谋
週洲%楊科%陳麗麗%陳虹亮%李忠玉%吳移謀
주주%양과%진려려%진홍량%리충옥%오이모
肺炎嗜衣原体%热休克蛋白10%Toll样受体%肿瘤坏死因子-α
肺炎嗜衣原體%熱休剋蛋白10%Toll樣受體%腫瘤壞死因子-α
폐염기의원체%열휴극단백10%Toll양수체%종류배사인자-α
Chlamydophila pneumoniae(Cpn)%Heat shock protein 10%Toll like receptor%Tumor necrosis factor-α
目的 探讨肺炎嗜衣原体(Cpn)热休克蛋白10(HSP10)诱导人单核细胞分泌炎症因子的作用及Toll样受体(TLR)2、TLR4与此作用的相关性.方法 制备Cpn HSP10(CHSP10)纯化蛋白,去内毒素活性后用不同浓度(0.5、1、5、10、20、30μ/ml)刺激THP-1细胞0、6、12、24、36、48、60 h,并比较蛋白不同处理组别中TNF-α的水平差异；以间接免疫荧光及RT-PCR鉴定THP-1细胞上的TLR4及TLR2；分离C3H系野生型和TLR4缺陷型小鼠巨噬细胞,以CHSP10刺激后检测TNF-α水平；用抗TLR2/TLR4单克隆抗体预孵育细胞,ELISA检测CHSP10刺激细胞前后TNF-α的变化.结果 CHSP10刺激THP-1细胞引起上清液炎症因子TNF-α水平显著增加,经加热等处理后蛋白诱生TNF-α的作用明显降低.THP-1细胞可检测到TLR2及TLR4的mRNA及蛋白表达,CHSP10诱生C3H系野生型小鼠细胞分泌的TNF-α明显高于TLR4缺陷型小鼠细胞,TLR2/4经单克隆抗体作用后均可显著降低CHSP10诱导THP-1分泌TNF-α的水平.结论 CHSP10可能作为炎症相关蛋白参与了Cpn对宿主细胞的致炎作用；并且TLR2及TLR4在该炎症刺激信号的传递过程中发挥一定的作用.
目的 探討肺炎嗜衣原體(Cpn)熱休剋蛋白10(HSP10)誘導人單覈細胞分泌炎癥因子的作用及Toll樣受體(TLR)2、TLR4與此作用的相關性.方法 製備Cpn HSP10(CHSP10)純化蛋白,去內毒素活性後用不同濃度(0.5、1、5、10、20、30μ/ml)刺激THP-1細胞0、6、12、24、36、48、60 h,併比較蛋白不同處理組彆中TNF-α的水平差異；以間接免疫熒光及RT-PCR鑒定THP-1細胞上的TLR4及TLR2；分離C3H繫野生型和TLR4缺陷型小鼠巨噬細胞,以CHSP10刺激後檢測TNF-α水平；用抗TLR2/TLR4單剋隆抗體預孵育細胞,ELISA檢測CHSP10刺激細胞前後TNF-α的變化.結果 CHSP10刺激THP-1細胞引起上清液炎癥因子TNF-α水平顯著增加,經加熱等處理後蛋白誘生TNF-α的作用明顯降低.THP-1細胞可檢測到TLR2及TLR4的mRNA及蛋白錶達,CHSP10誘生C3H繫野生型小鼠細胞分泌的TNF-α明顯高于TLR4缺陷型小鼠細胞,TLR2/4經單剋隆抗體作用後均可顯著降低CHSP10誘導THP-1分泌TNF-α的水平.結論 CHSP10可能作為炎癥相關蛋白參與瞭Cpn對宿主細胞的緻炎作用；併且TLR2及TLR4在該炎癥刺激信號的傳遞過程中髮揮一定的作用.
목적 탐토폐염기의원체(Cpn)열휴극단백10(HSP10)유도인단핵세포분비염증인자적작용급Toll양수체(TLR)2、TLR4여차작용적상관성.방법 제비Cpn HSP10(CHSP10)순화단백,거내독소활성후용불동농도(0.5、1、5、10、20、30μ/ml)자격THP-1세포0、6、12、24、36、48、60 h,병비교단백불동처리조별중TNF-α적수평차이；이간접면역형광급RT-PCR감정THP-1세포상적TLR4급TLR2；분리C3H계야생형화TLR4결함형소서거서세포,이CHSP10자격후검측TNF-α수평；용항TLR2/TLR4단극륭항체예부육세포,ELISA검측CHSP10자격세포전후TNF-α적변화.결과 CHSP10자격THP-1세포인기상청액염증인자TNF-α수평현저증가,경가열등처리후단백유생TNF-α적작용명현강저.THP-1세포가검측도TLR2급TLR4적mRNA급단백표체,CHSP10유생C3H계야생형소서세포분비적TNF-α명현고우TLR4결함형소서세포,TLR2/4경단극륭항체작용후균가현저강저CHSP10유도THP-1분비TNF-α적수평.결론 CHSP10가능작위염증상관단백삼여료Cpn대숙주세포적치염작용；병차TLR2급TLR4재해염증자격신호적전체과정중발휘일정적작용.
Objective To investigate the effect of heat shock protein 10 (HSP1O) of Chlamydophila pneumoniae in inducing TNF-α on THP-1 cells and the roles of TLR4 and TLR2 involved in it.Methods Purified native recombinant HSP10 from Cpn(CHSP10) were produced and inactivated the endotoxin contamination,then different concentration (0.5,1,5,10,20,30 μg/ml) of CHSP10 were used to stimulate THP-1 for different time (0,6,12,24,36,48,60 h).TNF-α were measured by using human TNF-α ELISA kit and compared among different groups.THP-1 were collected and analyzed for TLR2 and TLR4 mRNA levels and protein expression by RT-PCR and immunofluorescence.Peritoneal macrophages isolated from wide-type (C3 H/HeN) and TLR4-deficient mice (C3H/HeJ) were stimulated with endotoxin-free proteins respectively,and the TNF-α were measured.Furthermore,neutralizing anti-human TLR2/TLR4 McAb as a blocking Ab was preincubated with THP-1,after stimulation with CHSP10,ELISA was used to detect the concentration of TNF-α.Results TNF-α can be induced with CHSP10 in THP-1,while it significantly decreased with heated or deproteinized CHSP10.Both TLR2 and TLR4 mRNA and protein were detected in THP-1.Macrophages from C3H/HeN mice displayed higher TNF-α compared with it from C3H/HeJ mice after stimulation with CHSP10.The CHSP10-induced TNF-α would obviously decline when treated with antiTLR2/TLR4 McAb.Conclusion As a potential inflammation related protein,CHSP10 are involved in the pathogenesis of Cpn inducing inflammation cytokine TNF-α.TLR2 and TLR4 appear to be involved in CHSP10-mediated expression of TNF-α.
