CAJ | 학술논문

目的评价MTB Rv3671 c蛋白对人巨噬细胞(THP-1)的影响及机制.方法将编码MTB Rv3671c蛋白的基因克隆到pET-28a质粒,并在大肠埃希菌中进行表达,采用镍亲和层析和离子层析法纯化重组Rv3671c蛋白,Lowry法测定蛋白浓度,并用纯化蛋白刺激分化成熟巨噬细胞,采用Hochest染色法观察细胞的转归(凋亡及坏死)情况,同时取上清液用ELISA法检测TNF-α和IL-1β 的浓度.结果 MTB Rv3671c蛋白在大肠埃希菌中成功表达,层析纯化获得纯度为95.0％的重组Rv3671c蛋白,蛋白浓度可达0.4 mg/ml.Rv3671c蛋白刺激下经Hochest染色可见巨噬细胞核以坏死状多见,上清液中TNF-α和IL-13水平最高分别可达19 000、16 500 pg/ml；而未加蛋白干预组TNF-α和IL-1β水平最高仅为2100、3800 pg/ml,均明显低于干预组.结论 MTB Rv3671c蛋白可诱导人THP-1坏死,而该死亡可能与细胞因子TNF-α和IL-1β高表达有关.
목적 평개MTB Rv3671 c단백대인거서세포(THP-1)적영향급궤제.방법 장편마MTB Rv3671c단백적기인극륭도pET-28a질립,병재대장애희균중진행표체,채용얼친화층석화리자층석법순화중조Rv3671c단백,Lowry법측정단백농도,병용순화단백자격분화성숙거서세포,채용Hochest염색법관찰세포적전귀(조망급배사)정황,동시취상청액용ELISA법검측TNF-α화IL-1β 적농도.결과 MTB Rv3671c단백재대장애희균중성공표체,층석순화획득순도위95.0％적중조Rv3671c단백,단백농도가체0.4 mg/ml.Rv3671c단백자격하경Hochest염색가견거서세포핵이배사상다견,상청액중TNF-α화IL-13수평최고분별가체19 000、16 500 pg/ml；이미가단백간예조TNF-α화IL-1β수평최고부위2100、3800 pg/ml,균명현저우간예조.결론 MTB Rv3671c단백가유도인THP-1배사,이해사망가능여세포인자TNF-α화IL-1β고표체유관.
Objective To assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis (M.tuberculosis).Methods The gene encoding Rv3671 c protein of M.tuberculosis was cloned into pET-28a vector and then expressed in Escherichia coli.The Rv3671c was purified with Ni-NTA affinity and ion exchange chromatography.The detection of protein concentration was by Lowry method.THP-1 cell was stimulated with Rv3671c protein and cells were analyzed by Hochest staining under fluorescence microscopy to assay cell death (apoptosis and necrosis).TNF-α and IL-1β were detected by ELISA at each stimulating time.Results The Rv3671c protein of M.tuberculosis was successfully expressed in Escherichia coli.The purity of recombinant Rv3671c protein was 95％,and the protein concentration was up to 0.4 mg/ml.The nucleus of THP-1 was isolated and necrosis-like under fluorescence when cells were stimulated by Rv3671c protein.The levels of TNF-α and IL-1β in supernatant were 19 000 and 16 500 pg/ml respectively,and were significantly higher than control cells with the levels of 2100 and 3800 pg/ml separately.Conclusion The necrosis of THP-1 cells could be stimulated by Rv3671c protein of M.tuberculosis and it was probably associated with high cytokines TNF-α and IL-13 levels.