CAJ | 학술논문

目的研究肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)衣原体蛋白酶样活性因子(Chlamydial protease-like activity factor,CPAF)能否在体外诱导人单核细胞THP-1产生前炎症细胞因子和凋亡,为进一步探索Cpn感染宿主致病的分子机制提供实验依据.方法将Cpn CPAF全基因克隆于pGEX6p-2载体上,在大肠杆菌(E coli)中诱导表达重组蛋白,经去内毒素纯化柱和琼脂糖凝胶FF获得纯化且无脂多糖(LPS)污染的重组蛋白GST-CPAF.以不同浓度的该蛋白作用于THP-1,ELISA检测TNF-α吨和IL-6水平.MTT法检测经该蛋白处理后THP-1的增生或抑制作用,用Hoechst33258荧光染色、DNA片段化分析、Armexin V-FITC-PI染色法检测细胞凋亡情况.结果制备的莺组蛋白GST-CPAF以时间和剂量依赖方式刺激THP-1分泌TNF-α和IL-6,并以剂量依赖方式抑制THP-1增殖;当GST-CPAF刺激THP-1细胞24 h后,能诱导细胞调亡.结论制备的Cpn重组蛋白GST-CPAF能诱导THP-1产生前炎症细胞因子和凋亡,可能是Cpn致病机制中的一个因素.
목적 연구폐염기의원체(Chlamydophila pneumoniae,Cpn)의원체단백매양활성인자(Chlamydial protease-like activity factor,CPAF)능부재체외유도인단핵세포THP-1산생전염증세포인자화조망,위진일보탐색Cpn감염숙주치병적분자궤제제공실험의거.방법 장Cpn CPAF전기인극륭우pGEX6p-2재체상,재대장간균(E coli)중유도표체중조단백,경거내독소순화주화경지당응효FF획득순화차무지다당(LPS)오염적중조단백GST-CPAF.이불동농도적해단백작용우THP-1,ELISA검측TNF-α둔화IL-6수평.MTT법검측경해단백처리후THP-1적증생혹억제작용,용Hoechst33258형광염색、DNA편단화분석、Armexin V-FITC-PI염색법검측세포조망정황.결과 제비적앵조단백GST-CPAF이시간화제량의뢰방식자격THP-1분비TNF-α화IL-6,병이제량의뢰방식억제THP-1증식;당GST-CPAF자격THP-1세포24 h후,능유도세포조망.결론 제비적Cpn중조단백GST-CPAF능유도THP-1산생전염증세포인자화조망,가능시Cpn치병궤제중적일개인소.
Objective To express and purify Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,for investigating the effect of its recombinant protein GST-CPAF in inducing human monocytic cells to secrete proinflammatory cytokines and cell apoptosis.Methods The recom-bination expression plasmid pGEX6p-2/CPAF from Chlamydophila pneumoniae was transformed into E.coli.The recombination GST-CPAF was expressed after induction by IPTG,and purified by a agarose gel FF.Human monocytic cells were stimulated by the GST-CPAF to test the production of tumor necrosis factor a(TNF-α)and interleukin-6(IL- 6)by ELISA.Inhibition of cells proliferation with GST-CPAF was assessed by MTT.The THP-1 cell apoptosis stimulated by GST-CPAF was detected by Hoechst33258 fluorescence staining,DNA fragmentation analysis and cell apeptosis was detested bv Annexin V-FITC-propidiuum iodide (PI)staining.Results The recombination protein GST-CPAF was successfully expressed with high level in E.coli,and stimulated human monocytic cells to produce proinflammatory cytokines including TNF-α and IL-6 in a dose-and time-dependent manner.Otherwise,the GST-CPAF inhibited the growth of human monocytic cell in a dose-dependent manner.Apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy,DNA ladders in apoptosis cells were detected after 24 h with the GST-CPAF.Conclusion The GST-CPAF from Chlamydophila pneumoniae can induce the secretion of proinflammatory cytokines TNF-α and IL-6 by human monocytic cells,and inhibited the proliferation of THP-1 cell and apoptosis in vitro.