Abstract: Objective　To investigate the regulatory role of recombinant Trichinella spiralis cysteine protease inhibitors (rTs?Cys) in induction of polarization of bone marrow?derived macrophages (BMDMs) in vitro. Methods　BMDMs were captured and cultured in conditioned medium for 7 days. Then, mature BMDMs were harvested and assigned into four groups. Cells in Group A (negative control) were given 10 ng/mL IFN?γ combined with 100 ng/mL LPS, cells in Group B (positive control) were treated with IL?4 and IL?10 (at 10 ng/mL both), and cells in Group C (recombinant protein alone) were stimulated with 1 μg/mL rTs?Cys, while cells in Group D (protein co?culture) were simultaneously treated with 1 μg/mL rTs?Cys, 10 ng/mL IFN?γ and 100 ng/mL LPS. Cells and culture supernatant were collected 24 hour post?treatment, and the proportions of F4/80+, CD11b+, CD206+ and CD11c+ cells were detected by flow cytometry. The levels of interleukin IL?6 (IL?6), tumor necrosis factor?α (TNF?α), IL?10 and transforming growth factor?β (TGF?β) in the cell culture supernatant were measured by ELISA and the CD86+ and CD206+ phenotypes were identified by immunofluorescent staining. Results　Flow cytometry detected no significant difference in the proportion of F4/80+ CD11b+ CD11c+ cells among the four groups (F = 46.184, P < 0.001), and a lower proportion of F4/80+ CD11b+ CD11c+ cells was seen in groups C and D than in group A (all P values < 0.001). There was a significant difference in the proportion of F4/80+ CD11b+ CD206+ cells among the four groups (F = 11.032, P < 0.001), and a greater proportion of F4/80+ CD11b+ CD206+ cells was seen in groups C and D than in group A (all P values < 0.01). Immunofluorescent staining showed higher CD206+ expression and lower CD86+ expression in groups C and D than in Group A. There were significant differences in the IL?6 and (F = 3.950, P < 0.001) and TNF?α (F = 205.827, P < 0.001) levels in the cell culture supernatants among the four groups, and significantly lower IL?6 and TNF?α levels were measured in groups C and D than in Group A (both P < 0.05). There were significant differences in the IL?10 and (F = 8.274, P < 0.001) and TGF?β (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL?10 and TGF?β levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF?β level was significantly higher in Group D than in Group A (P < 0.05); however, there was no significant difference in the IL?10 level between groups D and A (P > 0.05). Conclusion　rTs?Cys may induce the polarization of BMDMs to anti?inflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.

Key words: Trichinella spiralis, Cysteine protease inhibitor, Macrophage, Immune regulation

摘要： 目的　探讨重组旋毛虫半胱氨酸蛋白酶抑制（rTs?Cys）对体外诱导骨髓来源巨噬细胞（BMDMs）极化的调控作用。方法　获取BMDMs并培养至条件培养基中，7 d后获取成熟BMDMs。将此BMDMs分为阴性对照组（A组）、阳性对照组（B组）、单独重组蛋白组（C组）和蛋白共培养组（D组），A组细胞给予10 ng/mL γ?干扰素（IFN?γ）和100 ng/mL 脂多糖（LPS）刺激，B组细胞给予10 ng/mL白细胞介素（IL）?4和10 ng/mL IL?10刺激，C组细胞给予1 μg/mL rTs?Cys干预，D组细胞给予1 μg/mL rTs?Cys、10 ng/mL IFN?γ及100 ng/mL LPS刺激。干预24 h后，收集细胞及培养上清，采用流式细胞术检测各组细胞中F4/80+、CD11b+、CD206+和CD11c+细胞比例，采用酶联免疫吸附试验（ELISA）检测细胞培养上清中IL?6、肿瘤坏死因子?α（TNF?α）、IL?10及转化生长因子?β（TGF?β）水平，采用免疫荧光染色鉴定CD86+、CD206+表型。结果　流式细胞术检测结果显示，4组F4/80+ CD11b+CD11c+细胞比例差异具有统计学意义（F = 46.184，P < 0.001），C组及D组F4/80+ CD11b+ CD11c+细胞比例均较A组显著降低（P均 < 0.001）；4组F4/80+ CD11b+ CD206+细胞比例差异具有统计学意义（F = 11.032，P < 0.01），C组及D组F4/80+ CD11b+ CD206+细胞比例均较A组显著升高（P均 < 0.01）。免疫荧光染色显示，C组及D组CD206+表达水平较A组显著上调，两组CD86+表达水平较A组下降。ELISA检测结果显示，4组细胞培养上清液中IL?6（F = 3.950，P < 0.001）和TNF?α水平（F = 205.827，P < 0.001）差异均有统计学意义；C组及D组IL?6和TNF?α水平均较A组显著下降（P均 < 0.05）。4组细胞培养上清液中IL?10（F =8.274，P < 0.001）和TGF?β水平（F = 13.559，P < 0.01）差异均有统计学意义；C组IL?10、TGF?β水平较A组显著增加（P均??0.05）。结论　rTs?Cys体外能诱导BMDMs向抗炎特性的M2方向极化，并抑制M1型巨噬细胞活化。

关键词: 旋毛虫, 半胱氨酸蛋白酶抑制剂, 巨噬细胞, 免疫调节