两种蠕虫半胱氨酸蛋白酶抑制剂对小鼠腹腔渗出细胞一氧化氮产生及细胞因子分泌的影响

发布时间：2019-03-08 20:39

【摘要】：目的观察两种蠕虫半胱氨酸蛋白酶抑制剂(cystatin)体外对小鼠腹腔渗出细胞(PEC)一氧化氮(NO)的产生及细胞因子分泌的影响。方法无痛处死BALB/c小鼠10只,收集腹腔内液体,制备渗出细胞(PEC),主要为巨噬细胞。将源自旋毛虫(Trichinella spiralis)及广州管圆线虫(Angiostrongylus cantonensis)的cystatin(Tscystatin,Accystatin)与脂多糖(LPS)刺激的小鼠PEC共孵育,实验分为RPMI组、LPS组、Accystatin+LPS组和Tscystatin+LPS组,除RPMI组外,其余3组均提前2 h用LPS(2μg/ml)刺激贴壁细胞制备炎症模型,Accystatin+LPS组和Tscystatin+LPS组分别用2μg/ml Accystatin或Tscystatin与LPS刺激后的细胞共孵育,每组6孔,培养24 h后,收集上清,用ELISA和硝酸还原酶法检测上清中α肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、IL-10等细胞因子和NO的水平。采用SPSS11.5统计学软件处理实验数据。结果ELISA检测结果显示:Accystatin+LPS组细胞上清中促炎因子TNF-α和IL-6分别为(507.50±66.32)和(1 440.49±77.25)pg/ml,较LPS组的(454.15±53.11)和(1 016.2±115.10)pg/ml明显升高(P0.05)。Tscystatin+LPS组分别为(296.35±55.30)和(793.54±86.61)pg/ml,较LPS组和Accystatin+LPS组明显降低(P0.05)。RPMI组和LPS组的IL-10分别为(38.80±6.71)和(53.66±7.72)pg/ml,差异无统计学意义(P0.05),Accystatin+LPS组和Tscystatin+LPS组的IL-10分别为(149.74±26.01)和(158.76±19.67)pg/ml,较LPS组均明显升高(P0.05)。Accystatin+LPS组和Tscystatin+LPS组NO水平分别为(12.54±2.12)和(7.95±1.40)μmol/L,较LPS组的(20.18±3.99)μmol/L均明显降低(P0.05),且Tscystatin+LPS组NO水平低于Accystatin+LPS组(P0.05)。结论旋毛虫和广州管圆线虫的cystatin均可抑制小鼠腹腔渗出细胞NO释放、上调IL-10的水平,Accystatin明显促进TNF-α及IL-6的分泌,Tscystatin则明显抑制两种细胞因子的水平。
[Abstract]:Aim to observe the effects of two cysteine protease inhibitors, (cystatin), on the production of nitric oxide (NO) and cytokine secretion in mouse peritoneal exudates (PEC) in vitro. Methods Ten BALB/c mice were killed painlessly. The peritoneal fluid was collected and the exudative cells (PEC), were mainly macrophages. Cystatin (Tscystatin,Accystatin), derived from Trichinella spiralis (Trichinella spiralis) and (Angiostrongylus cantonensis) of Angiostrongylus cantonensis, was incubated with PEC stimulated by lipopolysaccharide (LPS). The experiment was divided into three groups: RPMI group, LPS group, Accystatin LPS group and Tscystatin LPS group, except RPMI group. The other three groups were stimulated with LPS (2 渭 g / ml) 2 h earlier to prepare inflammatory model of, Accystatin LPS group and Tscystatin LPS group with 2 渭 g / ml Accystatin or Tscystatin co-incubated with LPS stimulated cells, respectively. The supernatant was collected after 24 h culture, and the cells in each group were incubated with 2 渭 g / ml Accystatin or Tscystatin for 24 h. The levels of tumor necrosis factor 伪 (TNF- 伪), interleukin 6 (IL-6), IL-10 and NO in supernatant were detected by ELISA and nitrate reductase. SPSS11.5 statistical software was used to process the experimental data. Results the results of ELISA showed that TNF- 伪 and IL-6 in the supernatant of: Accystatin LPS group were (507.50 卤66.32) pg/ml, and (1 440.49 卤77.25) pg/ml, respectively. The levels of (454.15 卤53.11) and (1 016.2 卤115.10) pg/ml in LPS group were significantly higher than those in). Tscystatin LPS group (296.35 卤55.30) and (793.54 卤86.61) pg/ml, respectively. Compared with LPS group and Accystatin LPS group, the IL-10 of). RPMI group and LPS group were (38.80 卤6.71) and (53.66 卤7.72) pg/ml, respectively (P0.05). The IL-10 of Accystatin LPS group and Tscystatin LPS group were (149.74 卤26.01) and (158.76 卤19.67) pg/ml, respectively. NO levels in). Accystatin LPS group and Tscystatin LPS group were significantly higher than those in LPS group (P 0.05 卤2.12) and (7.95 卤1.40) 渭 mol/L, vs (20.18 卤3.99) 渭 mol/L in LPS group (P0.05). The level of NO in Tscystatin LPS group was lower than that in Accystatin LPS group (P0.05). Conclusion both cystatin of Trichinella spiralis and Angiostrongylus cantonensis could inhibit the release of NO and up-regulate the level of IL-10 in peritoneal exudates of mice. Accystatin significantly promoted the secretion of TNF- 伪 and IL-6, while Tscystatin significantly inhibited the levels of both cytokines.
【作者单位】：蚌埠医学院病原生物学教研室安徽省感染与免疫重点实验室;蚌埠医学院组织学与胚胎学教研室;江南大学无锡医学院病原感染与免疫组;
【基金】：安徽省教育厅重点资助项目(No.KJ2017A235) 安徽省自然科学基金(No.1508085QH158) 安徽高校科研创新平台团队项目(No.2016-40) 安徽省高校优秀青年人才支持计划重点项目(No.gxyq ZD2016159) 国家大学生创新创业训练计划项目(No.201610367009)~~
【分类号】：R392

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