猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用

科研成果详情

题名	猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用
其他题名	Activation of polysaccharides from Polyporus umbellatus(Pers.) Fries on peritoneal macrophages from mice through Toll-like receptor 4
作者	许文1; 李心群2
发表日期	2010-08-15
发表期刊	中国药理学与毒理学杂志影响因子和分区
语种	中文
原始文献类型	学术期刊
关键词	猪苓多糖腹腔巨噬细胞 Toll样受体4
摘要	目的观察猪苓多糖(PPS)对小鼠腹腔巨噬细胞的活化作用,并研究Toll样受体4(TLR4)在其中的作用。方法用PPS12.5,25,50及100mg·L-1刺激C3H/HeN小鼠和TLR4缺陷的C3H/HeJ小鼠脾细胞72h或腹腔巨噬细胞24h,以[3H]TdR掺入法检测脾细胞增殖反应;以Griess法测定腹腔巨噬细胞培养上清一氧化氮(NO)水平,ELISA检测白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)含量;以溴化氰活化多糖的方法制备荧光胺(Flu)标记的PPS(Flu-PPS)及Flu-葡聚糖,应用流式细胞仪及激光共聚焦显微镜检测Flu-PPS与小鼠腹腔巨噬细胞的结合。结果 PPS可明显促进C3H/HeN小鼠脾细胞增殖并诱导腹腔巨噬细胞分泌NO,IL-1β和TNF-α,且具有浓度依赖性(r=0.999,P<0.01)。用抗TLR4单抗20mg·L-1与C3H/HeN小鼠腹腔巨噬细胞预孵育1h后加入PPS50mg·L-1,与单独PPS50mg·L-1孵育比较,巨噬细胞培养上清IL-1β和TNF-α浓度分别由(0.38±0.06)μg·L-1和(0.29±0.05)μg·L-1降至(0.18±0.01)μg·L-1和(0.12±0.02)μg·L-1,降幅达52.6%和58.6%(P<0.01)。PPS对C3H/HeN小鼠的脾细胞增殖、腹腔巨噬细胞产生IL-1β和TNF-α的作用明显强于C3H/HeJ小鼠:PPS12.5～100mg·L-1组脾细胞增殖分别增加了2.4,1.7,1.5和22.2倍,IL-1β增加了0.9,1.3,1.2和1.1倍,TNF-α增加了1.3,0.9,0.6和0.6倍,差异均有统计学意义(P<0.01)。流式细胞仪及激光共聚焦显微镜分析结果表明,Flu-PPS1mg·L-1与小鼠腹腔巨噬细胞结合的荧光强度显著高于Flu-葡聚糖,增加Flu-PPS浓度至5mg·L-1,荧光强度并未相应增强,表明Flu-PPS与小鼠腹腔巨噬细胞的结合具有饱和性;未标记的PPS100mg·L-1及抗TLR4单抗200mg·L-1可明显阻断Flu-PPS与巨噬细胞的结合。结论 PPS可能通过TLR4活化小鼠腹腔巨噬细胞。
其他摘要	OBJECTIVE To observe the effect of polysaccharides from Polyporus umbellatus(Pers.) Fries(PPS) on mouse peritoneal macrophages and evaluate the role of the Toll-like receptor(TLR) in the process. METHODS Splenocytes and peritoneal macrophages from C3H/HeN or C3H/HeJ mice were incubated with different concentrations of PPS(12.5,25,50 and 100 mg·L~(-1)) for 72 h or 24 h. Splenocyte proliferation was analyzed with [~3H]TdR incorporation method. Nitric oxide(NO) was measured by Griess method and cytokines of culture supernatants were detected by enzyme linked immunosorbent assay(ELISA).The fluoresceinamine-labeled PPS(Flu-PPS) and dextran(Flu-dextran) were prepared by the cyanogen bromide activation method. The cell-binding activity of Flu-PPS was analyzed with FACS and the confocal microscopy. RESULTS PPS significantly stimulated the proliferation of splenocytes from C3H/HeN mice and induced the production of NO, interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) of peritoneal macrophages in a concentration-dependent manner(r = 0.999,P < 0.01). Compared with the PPS 50 mg·L~(-1) group, the levels of IL-1β and TNF-α decreased from(0.38 ± 0.06) μg·L~(-1) and(0.29 ±0.05) μg·L~(-1) to(0.18 ±0.01) μg·L~(-1) and(0.12 ±0.02) μg·L~(-1) when macrophages from C3H/HeN mice were pretreated with anti-mouse TLR4 20 mg·L~(-1) monoclonal antibody for 1 h before adding PPS;the inhibitory rate was 52.6% and 58.6%(P < 0.05), respectively. The effect of PPS on splenocyte proliferation,NO,IL-1β and TNF-α production in C3H/HeN mice were stronger than in C3H/HeJ mice:splenocyte proliferation was increased 2.4,1.7,1.5 and 22.2 times, and IL-1β production increased 0.9,1.3,1.2 and 1.1 times,TNF-α production increased 1.3,0.9,0.6 and 0.6 times in PPS 12.5-100 mg·L~(-1) groups, respectively. The Flu intensity of murine macrophages staining with Flu-PPS(1 mg·L~(-1)) was higher than that of Flu-dextran group in FACS and confocal laser scanning microscopy analysis. The Flu intensity did not change while the concentration of PPS was increased to 5 mg·L~(-1), which showed affinity saturation in the macrophage surface with Flu-PPS. Unlabeled PPS(100 mg·L~(-1)) and anti-mouse TLR4 monoclonal antibody(200 mg · L~(-1)) significantly inhibited Flu-PPS binding with macrophages. CONCLUSION PPS may activate macrophages via TLR4
资助项目	温州市科学技术局资助项目（Y20080126）;温州医学院科研启动基金资助项目（QTJ07021）~~
ISSN	1000-3002
卷号	24 期号:04 页码:266-273
页数	8
收录类别	CNKI ; 北大核心 ; CSCD
学科领域	医药、卫生 ; 中国医学 ; 药学
URL	查看原文
CSCD记录号	CSCD:3975332
引用统计
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/70767
专题	附属第一医院基础医学院（机能实验教学中心）基础医学院（机能实验教学中心）_病原生物学与免疫学系
作者单位	1.温州医学院微生物学与免疫学教研室; 2.温州医学院附属第一医院急诊科
第一作者单位	病原生物学与免疫学系
第一作者的第一单位	病原生物学与免疫学系
推荐引用方式 GB/T 7714	许文,李心群. 猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用[J]. 中国药理学与毒理学杂志,2010,24(04):266-273.
APA	许文, & 李心群. (2010). 猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用. 中国药理学与毒理学杂志, 24(04), 266-273.
MLA	许文,et al."猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用".中国药理学与毒理学杂志 24.04(2010):266-273.