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目的:观察草珊瑚多糖对巨噬细胞RAW264.7体外释放一氧化氮,膜表面分子和共刺激分子的影响和细胞因子mRNA的影响。方法:实验分为空白对照组,γ干扰素(IFN-γ)诱导的模型组,草珊瑚多糖低、中、高剂量组,浓度分别为25,50,100 mg·L-1。药物作用于RAW264.7巨噬细胞株24 h后,用流式细胞术检测RAW264.7膜分子CD16/32,CD40,CD14,Toll样受体4(TIR4)的表达,实时荧光定量PCR(RT-PCR)检测炎症因子mRNA表达量;巨噬细胞诱导为M1后给予草珊瑚多糖干预,检测膜蛋白表达量和mRNA表达的差异,Griess方法检测一氧化氮释放的影响。结果:草珊瑚多糖可以有效的增加巨噬细胞RAW264.7膜蛋白CD16/32的表达,由空白的31.0±1.2上升到81.5±3,CD40由空白的5.2±0.9上升到81.5±2.6,CD14 28.5±1.6上升到86.7±5.4,TLR4的表达。对极化为M1亚型的巨噬细胞膜蛋白没有影响;草珊瑚多糖可以提高白细胞介素1β(IL-1β),IL-10,肿瘤坏死因子α(TNF-α),诱导型一氧化氮合成酶(iNOS)mRNA表达,而且存在剂量依赖性(P<0.01),能提高M1亚型的因子mRNA的表达。能提高巨噬细胞一氧化氮的分泌。结论:草珊瑚多糖显著促进巨噬细胞相关免疫分子的表达,提高相关免疫因子RNA的表达,调节巨噬细胞促炎和抗炎性细胞因子的表达,对免疫平衡功能的调节具有潜在的应用价值。
OBJECTIVE: To observe the effect of polysaccharides from S.corella on the release of nitric oxide, membrane surface molecules and costimulatory molecules in macrophage RAW264.7 in vitro and the effect of cytokines mRNA. Methods: The experiment was divided into blank control group, model group induced by IFN-γ and low, medium and high doses of Scutellaria Coralis polysaccharide. The concentrations were 25, 50 and 100 mg · L-1, respectively. After treated with RAW264.7 macrophages for 24 h, the expressions of CD16 / 32, CD40, CD14 and Toll-like receptor 4 (TIR4) in RAW264.7 cells were detected by flow cytometry. Real-time fluorescence quantitative PCR -PCR) was used to detect the mRNA expression of inflammatory cytokines. After induced by M1, macrophages were treated with polysaccharides of Artemisia grosvenorii to detect the difference of membrane protein expression and mRNA expression. Griess method was used to detect the release of nitric oxide. Results: Sarcandra polysaccharide could effectively increase the expression of macrophage RAW264.7 membrane protein CD16 / 32, from blank 31.0 ± 1.2 to 81.5 ± 3, CD40 up to 81.5 ± 2.6 from blank 5.2 ± 0.9, CD14 28.5 ± 1.6 to 86.7 ± 5.4, TLR4 expression. There was no effect on the membrane protein of macrophages polarized to M1 subtype. Scutellaria polysaccharides could increase IL-1β, IL-10, TNF-α, inducible nitric oxide synthase (INOS) mRNA in a dose-dependent manner (P <0.01), which can increase the mRNA expression of M1 subtype. Can improve macrophage nitric oxide secretion. CONCLUSION: Coral polysaccharide can significantly promote the expression of macrophage-associated immune molecules, enhance the expression of related immune factors RNA, regulate the expression of pro-inflammatory and anti-inflammatory cytokines in macrophages, and have potential value in the regulation of immune balance .