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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(P<0.05)。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(P<0.05)。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(P>0.05),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(P<0.05)。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(P<0.05),且Tscystatin+LPS组NO水平低于Accystatin+LPS组(P<0.05)。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。
Objective To investigate the effects of cystatin on nitric oxide (NO) production and cytokine secretion in mouse peritoneal exudate cells (PEC) in vitro. Methods Totally 10 BALB / c mice were killed without pain, and the peritoneal fluid was collected to produce exudative cells (PECs), mainly macrophages. The cystatin (Tscystatin, Accystatin) derived from Trichinella spiralis and Angiostrongylus cantonensis were co-incubated with lipopolysaccharide (LPS) -stimulated PEC. The experiment was divided into three groups: RPMI group, LPS group, Accystatin + LPS group and Tscystatin + LPS group. Except RPMI group, the other 3 groups were stimulated with LPS (2μg / ml) 2 hours before adherent cells to prepare inflammatory model. Accystatin + LPS group and Tscystatin + LPS group were treated with 2μg / ml Accystatin Or Tscystatin were incubated with LPS stimulated cells, each well was 6 wells, the supernatant was collected after 24 h of culture, and the levels of TNF-α, IL- 6 (IL-6), IL-10 and other cytokines and NO levels. SPSS11.5 statistical software was used to process the experimental data. Results The results of ELISA showed that the levels of pro-inflammatory cytokines TNF-α and IL-6 in the supernatants of Accystatin + LPS group were (507.50 ± 66.32) and (1 440.49 ± 77.25) pg / ml, ) And (1 016.2 ± 115.10) pg / ml were significantly increased (P <0.05). (296.35 ± 55.30) and (793.54 ± 86.61) pg / ml in Tscystatin + LPS group, which were significantly lower than those in LPS group and Accystatin + LPS group (P <0.05). IL-10 in RPMI group and LPS group were (38.80 ± 6.71) and (53.66 ± 7.72) pg / ml respectively, with no significant difference (P> 0.05). IL-10 in Accystatin + LPS group and Tscystatin + (149.74 ± 26.01) and (158.76 ± 19.67) pg / ml, respectively, which were significantly higher than those in LPS group (P <0.05). The NO levels in Accystatin + LPS group and Tscystatin + LPS group were (12.54 ± 2.12) and (7.95 ± 1.40) μmol / L, respectively, which were significantly lower than those in LPS group (20.18 ± 3.99) μmol / L The NO level in Tscystatin + LPS group was lower than that in Accystatin + LPS group (P <0.05). Conclusion Cystatin of Trichinella spiralis and Acremonium cercariae can inhibit NO release in peritoneal exudate cells and up-regulate the level of IL-10. Accystatin can significantly promote the secretion of TNF-α and IL-6. Tscystatin significantly inhibits the expression of two cytokines s level.