不同毒力结核杆菌的纯化蛋白质衍生物对人巨噬细胞凋亡的诱导及其与Toll样受体-2的相关性

目的 了解两种不同毒力结核杆菌的纯化蛋白衍生物刺激下人单核-巨噬细胞(THP-1)凋亡的差异及其与Toll样受体-2(TLR-2)的相关性.方法 分别用等浓度的高毒力结核杆菌衍生蛋白(H37Rv-PPD)和低毒力结核杆菌衍生蛋白(BCG-PPD)在3、8、15和24 h刺激分化成熟的THP-1细胞;Hochest染色后观察细胞凋亡及坏死情况.流式细胞仪检测细胞Annexin V蛋白以判定凋亡及TLR-2表达情况;加入TLR-2阻断剂后,再用流式细胞仪测定细胞表面TLR-2的阻断效果及凋亡情况.结果 BCG-PPD刺激下细胞以凋亡多见,而H37Rv-PPD刺激下细胞核则多呈坏死状.凋亡率随时间升高,第24小时凋亡比例高达30.2%,而同时间点TLR-2的比例为8.8%;应用TLR-2阻断剂后,每个时间点TLR-2表达比例均在3%以下,对应时间点凋亡比例下降,第24小时凋亡比例仅为10.5%.H37Rv-PPD可引起TLR-2高表达,第24小时TLR-2表达率为17.2%,该时间点凋亡率仅为7.7%;TLR-2阻断后,其表达率在对照波动范围内,但凋亡率与TLR-2未阻断前相比变化不大.结论 BCG-PPD主要诱导THP-1的凋亡,且与TLR-2有一定的相关性;而H37Rv-PPD主要诱导THP-1的坏死.

Abstract：

Objective To study the cell death in macrophages (THP-1) stimulated with different agonists (H37Rv-PPD or BCG-PPD) and to investigate the relationship between Toll like receptor (TLR)-2 and THP-1 apoptosis. Methods H37Rv-PPD and BCG-PPD were used to stimulate THP-1 cells for 3 h, 8 h, 15 h and 24 h, respectively with or without TLR-2 blockade. Cells were analyzed by flow cytometry to detect the TLR-2 expression. Annexin V staining and Hochest staining were performed to evaluate apoptosis. Results The apoptosis cells were increased when stimulated with BCG-PPD and the percentage was 30.2% at 24 h, which were confirmed by Hochest staining.However, the expression of TLR-2 did not increase simultaneously with percentage of 8.8% at 24 h.Nevertheless, most cells presented with necrosis form when stimulated with H37Rv-PPD and the expression of TLR-2 remained at high level with the percentage of 17.2% at 24 h, while the percent of apoptosis rate was only 7.7%. Under treatment of TLR-2 antibodies, the percentage of apoptosis decreased to 10.5% at 24 h of BCG-PPD stimulation and TLR-2 expressions were down-regulated to less than 3% at all time points; but after H37Rv-PPD stimulation, the percentage of apoptosis and TLR-2 expression did not changed obviously. Conclusions The attenuated BCG-PPD induces THP-1 apoptosis predominately, which is partially correlated with TLR-2 expression. While virulent H37Rv-PPD induces THP-1 necrosis predominately.

Purified protein derivatives tuberculin from virulent or attenuated strains of Mycobacterium tuberculosis induces cell death of human macrophages in different manner

Objective To study the cell death in macrophages (THP-1) stimulated with different agonists (H37Rv-PPD or BCG-PPD) and to investigate the relationship between Toll like receptor (TLR)-2 and THP-1 apoptosis. Methods H37Rv-PPD and BCG-PPD were used to stimulate THP-1 cells for 3 h, 8 h, 15 h and 24 h, respectively with or without TLR-2 blockade. Cells were analyzed by flow cytometry to detect the TLR-2 expression. Annexin V staining and Hochest staining were performed to evaluate apoptosis. Results The apoptosis cells were increased when stimulated with BCG-PPD and the percentage was 30.2% at 24 h, which were confirmed by Hochest staining.However, the expression of TLR-2 did not increase simultaneously with percentage of 8.8% at 24 h.Nevertheless, most cells presented with necrosis form when stimulated with H37Rv-PPD and the expression of TLR-2 remained at high level with the percentage of 17.2% at 24 h, while the percent of apoptosis rate was only 7.7%. Under treatment of TLR-2 antibodies, the percentage of apoptosis decreased to 10.5% at 24 h of BCG-PPD stimulation and TLR-2 expressions were down-regulated to less than 3% at all time points; but after H37Rv-PPD stimulation, the percentage of apoptosis and TLR-2 expression did not changed obviously. Conclusions The attenuated BCG-PPD induces THP-1 apoptosis predominately, which is partially correlated with TLR-2 expression. While virulent H37Rv-PPD induces THP-1 necrosis predominately.