【摘要】：目的觀察兩種蠕蟲半胱氨酸蛋白酶抑制劑(cystatin)體外對(duì)小鼠腹腔滲出細(xì)胞(PEC)一氧化氮(NO)的產(chǎn)生及細(xì)胞因子分泌的影響。方法無(wú)痛處死BALB/c小鼠10只,收集腹腔內(nèi)液體,制備滲出細(xì)胞(PEC),主要為巨噬細(xì)胞。將源自旋毛蟲(Trichinella spiralis)及廣州管圓線蟲(Angiostrongylus cantonensis)的cystatin(Tscystatin,Accystatin)與脂多糖(LPS)刺激的小鼠PEC共孵育,實(shí)驗(yàn)分為RPMI組、LPS組、Accystatin+LPS組和Tscystatin+LPS組,除RPMI組外,其余3組均提前2 h用LPS(2μg/ml)刺激貼壁細(xì)胞制備炎癥模型,Accystatin+LPS組和Tscystatin+LPS組分別用2μg/ml Accystatin或Tscystatin與LPS刺激后的細(xì)胞共孵育,每組6孔,培養(yǎng)24 h后,收集上清,用ELISA和硝酸還原酶法檢測(cè)上清中α腫瘤壞死因子(TNF-α)、白細(xì)胞介素-6(IL-6)、IL-10等細(xì)胞因子和NO的水平。采用SPSS11.5統(tǒng)計(jì)學(xué)軟件處理實(shí)驗(yàn)數(shù)據(jù)。結(jié)果ELISA檢測(cè)結(jié)果顯示:Accystatin+LPS組細(xì)胞上清中促炎因子TNF-α和IL-6分別為(507.50±66.32)和(1 440.49±77.25)pg/ml,較LPS組的(454.15±53.11)和(1 016.2±115.10)pg/ml明顯升高(P0.05)。Tscystatin+LPS組分別為(296.35±55.30)和(793.54±86.61)pg/ml,較LPS組和Accystatin+LPS組明顯降低(P0.05)。RPMI組和LPS組的IL-10分別為(38.80±6.71)和(53.66±7.72)pg/ml,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),Accystatin+LPS組和Tscystatin+LPS組的IL-10分別為(149.74±26.01)和(158.76±19.67)pg/ml,較LPS組均明顯升高(P0.05)。Accystatin+LPS組和Tscystatin+LPS組NO水平分別為(12.54±2.12)和(7.95±1.40)μmol/L,較LPS組的(20.18±3.99)μmol/L均明顯降低(P0.05),且Tscystatin+LPS組NO水平低于Accystatin+LPS組(P0.05)。結(jié)論旋毛蟲和廣州管圓線蟲的cystatin均可抑制小鼠腹腔滲出細(xì)胞NO釋放、上調(diào)IL-10的水平,Accystatin明顯促進(jìn)TNF-α及IL-6的分泌,Tscystatin則明顯抑制兩種細(xì)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 偽 (TNF- 偽), 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- 偽 and IL-6 in the supernatant of: Accystatin LPS group were (507.50 鹵66.32) pg/ml, and (1 440.49 鹵77.25) pg/ml, respectively. The levels of (454.15 鹵53.11) and (1 016.2 鹵115.10) pg/ml in LPS group were significantly higher than those in). Tscystatin LPS group (296.35 鹵55.30) and (793.54 鹵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 鹵6.71) and (53.66 鹵7.72) pg/ml, respectively (P0.05). The IL-10 of Accystatin LPS group and Tscystatin LPS group were (149.74 鹵26.01) and (158.76 鹵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 鹵2.12) and (7.95 鹵1.40) 渭 mol/L, vs (20.18 鹵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- 偽 and IL-6, while Tscystatin significantly inhibited the levels of both cytokines.
【作者單位】： 蚌埠醫(yī)學(xué)院病原生物學(xué)教研室 安徽省感染與免疫重點(diǎn)實(shí)驗(yàn)室;蚌埠醫(yī)學(xué)院組織學(xué)與胚胎學(xué)教研室;江南大學(xué)無(wú)錫醫(yī)學(xué)院病原感染與免疫組;
【基金】：安徽省教育廳重點(diǎn)資助項(xiàng)目(No.KJ2017A235) 安徽省自然科學(xué)基金(No.1508085QH158) 安徽高�？蒲袆�(chuàng)新平臺(tái)團(tuán)隊(duì)項(xiàng)目(No.2016-40) 安徽省高校優(yōu)秀青年人才支持計(jì)劃重點(diǎn)項(xiàng)目(No.gxyq ZD2016159) 國(guó)家大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(No.201610367009)~~
【分類號(hào)】：R392

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