神经介素U在体外对猪树突细胞活性及功能的影响

Effects of neuromedin U on activity and function of porcine dendritic cells in vitro

[目的]本文旨在探讨不同浓度神经介素U(NMU)对外周血单核源树突细胞(DC)刺激T淋巴细胞增殖和相关细胞因子分泌的影响。[方法]采集小梅山猪外周血,用集落刺激因子(GM-CSF)和IL-4联合诱导外周血单核细胞形成未成熟树突细胞,再加入脂多糖(LPS)刺激获得成熟的树突细胞,同时观察DC形态。加入不同浓度(0.01、0.1、1、10、100、1 000 nmol·L^(-1))NMU并分别培养2、4、8和12 h后,收集细胞上清液,用ELISA法测定IL-4、IL-5和IL^(-1)3的浓度。加入不同浓度(0.1、1、10、100、1 000 nmol·L^(-1))NMU培养24 h后,收集细胞,用CCK-8试剂盒和细胞凋亡检测试剂盒分别检测DC刺激混合淋巴细胞时细胞的增殖和DC凋亡率。[结果]NMU(0.1~100 nmol·L^(-1))能够抑制未成熟树突细胞(i DC)的细胞凋亡(P<0.01),且NMU与NMU+LPS作用效果相一致,10 nmol·L^(-1)NMU组与10 nmol·L^(-1)NMU+LPS组都能降低i DC凋亡,凋亡率分别为2.383%和2.360%,与LPS组相比,NMU+LPS组抑制i DC细胞凋亡效果极显著(P<0.01),说明NMU与LPS协同发挥抑制i DC细胞凋亡作用;与对照组相比,0.1~100 nmol·L^(-1)NMU能极显著促进m DC分泌IL-5(P<0.01)和抑制IL-4分泌(P<0.01)。NMU对IL^(-1)3的影响呈现多样性,低剂量(0.01~0.1 nmol·L^(-1))NMU在2、4 h抑制m DC细胞分泌IL^(-1)3(P<0.05),中剂量(1~10 nmol·L^(-1))NMU在2 h先抑制m DC分泌IL^(-1)3(P<0.05),4 h后又促进其分泌(P<0.05),高剂量(100~1 000 nmol·L^(-1))NMU则促进m DC分泌IL^(-1)3(P<0.05)。经NMU诱导后,i DC和m DC均能促进T淋巴细胞增殖(P<0.01)。[结论]在一定浓度范围内,NMU能够抑制猪树突细胞的凋亡,并提高其细胞活性和促进树突细胞刺激淋巴细胞增殖的功能,并能影响树突细胞细胞因子分泌,提示神经介素U参与了对猪免疫功能的调节。

[ Objectives ] The paper aims to explore the efficiency of neuromedin U （NMU）with different concentrations treatments on the cytokine secretion and T lymphocyte proliferation by pig dendritic ceils （DC）. [ Methods ] In this study,immature dendritic cells （iDC） were originated from pig peripheral blood monocytes cultured with GM-CSF and IL-4, and then triggered DC maturation with LPS. Varied doses of NMU（0.01,0.1,1,10,100,1 000 nmol. L-1） were added into iDC and mature dendritic cells （mDC）at different time points（2,4,8, 12 h）,then the culture supernatant of mDC was collected to detect IL-4,IL-5 and IL-13 by ELISA kit. Finally, the apoptosis of DC and the capacity of DC to evaluate T cell proliferation were determined by apoptosis detection kit and CCK-8 kit. [ Results ] NMU （0.1-100 nmol. L-l） can inhibit the apoptosis of iDC （2.383%）（ P〈0.01 ）, which was consistent with the effect of NMU ＋LPS （2.360%）. Furthermore, compared with LPS, NMU＋LPS can significantly inhibit the apoptosis of iDC, which suggested that NMU had a synergistic effect with LPS in inhibiting apoptosis of iDC. Compared with control group, NMU （0. 1- 100 nmol＂ L-1） can significantly increase the expression of IL-5 （ P〈 0.01 ） and reduce the expression of IL-4 （ P 〈 0.01 ）. However, the secretion of IL- 13 displayed a diversity trend. It turned out that low NMU（O.O1-0.1 nmol.L-~ ）can inhibit the expression of IL-13 in mDC during 2 h to 4 h（P〈0.05） ,and medium dose（ 1-10 nmol. L-1） NMU can inhibit the IL-13 release within 2 hours while it can increase the IL-13 secretion after 4 hours （P〈0.05）. High dose （ 100 - 1 000 nmol＂ L-~ ） NMU increased the expression of IL- 13 （ P〈0.05 ）. Both iDC and mDC can promote the proliferation of T lymphocytes after incubation with NMU （P〈0.01）. [ Conclusions] NMU has a remarked immunity effect. Its mechanism is probably based on promotion of the activity of DC and inhibition of the apoptosis of dendritic cells. Within the certain range of concentration, NMU can inhibit the apoptosis of porcine dendritic cells and increase the activity of DC, which enhance proliferation of T cells and alter the cytokine secretion of mDC.