野生亚侧耳多糖的提取和对巨噬细胞Raw264.7的免疫调节作用研究

Extraction of Polysaccharides from Wild Hohenbuehelia serotina and Its Immunomodulatory Function in Raw264.7 Cells

本研究从亚侧耳子实体中分离出水溶性亚侧耳多糖(HSP),通过体外实验评估其对Raw264.7巨噬细胞的免疫调节作用。采用WST-1法检测HSP对巨噬细胞增殖作用的影响,Griess试剂检测一氧化氮(NO)浓度,ELISA法检测HSP对淋巴细胞中一肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和前列腺素E2(prostaglandins E2,PGE2)等细胞因子分泌量的影响,反转录聚合酶链式反应分析诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)、白细胞介素-6(IL-6)、TNF-α和IL-1β的转录水平表达量。结果表明,HSP可显著提高Raw264.7细胞内NO的浓度,在HSP浓度为100.00μg/m L时,NO的分泌量达到最大值(29.94 mmol/L)。与空白组相比,HSP可以显著提高TNF-α、IL-1β、PGE2等免疫因子(P<0.05)的分泌量,其中IL-1β、PGE2的25μg/m L剂量组中各因子分泌量(31.9524 pg/m L,1.8174 ng/m L)均与LPS(脂多糖)阳性对照组相接近。反转录聚合酶链式反应分析结果表明,HSP能够提高iNOS和COX-2的蛋白表达量。HSP具有免疫调节的功能,可以作为免疫调节剂添加到功能性食品和药品中。

Water soluble Hohenbuehelia serotina polysaccharides( HSP) was isolated and its immunomodulatory activities in macrophage Raw264.7 cells were evaluated in vitro.Macrophage proliferation was conducted by WST-1.NO concentration was analyzed by griess reagent.Tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and prostaglandins E2( PGE2) was detected by ELISA.And transcriptional level of iNOS,COX-2,IL-6,TNF-α and IL-1β was determined by RT-qPCR. The results indicated that HSP could significantly stimulate intracellular nitric oxide( NO) concentration in Raw264.7 cells. When the concentration of HSP was 100.00 μg/m L,the secretion of NO reached to the maximum which was 30.4335 μmol/L.Compared with blank control,HSP could significantly increase cytokines in Raw264.7 cells such as the tumor necrosis factor-α( TNF-α),prostaglandin E2( PGE2) and interleukin-1β( IL-1β) levels,and its secretion of high dose group( 29.94 pg/m L,1.8174 ng/m L) was similar to that in the LPS positive control group. The results of RT-q PCR analysis showed that HSP also could enhance inducible nitric oxide synthase( iNOS) and cyclooxygenase-2( COX-2) expression in Raw264.7 cells. These results suggested that HSP had potential immunomodulating activities and could be applied in functional food and medicine.