红毛藻多糖的化学组成及其体外免疫诱导活性研究

目的:对红毛藻中多糖组分进行提取、分离纯化,分析其化学组成和体外免疫诱导活性,阐明红毛藻具有提高免疫力食药用功效机制,为红毛藻的高值化利用提供科学依据。方法:通过水提醇沉法从红毛藻中提取红毛藻粗多糖,经阳离子交换柱层析和Sephadex G75柱层析纯化得到红毛藻多糖(BFP),再用DEAE-cellulose52柱层析对BFP进行分级纯化得到3个多糖组分F1、F2和F3;采用PMP柱前衍生、HPLC及化学方法分析其单糖组成和化学成分;通过RAW264.7细胞模型分析BFP、F1、F2和F3的体外免疫诱导活性和其相关细胞信号传导通路。结果:组成分析结果表明BFP、F1、F2和F3均为杂多糖且单糖组成存在较大差异。细胞模型结果表明,BFP、F1、F2和F3在所测定质量浓度范围(0~100μg/mL)均显著诱导RAW264.7细胞活化,释放NO和分泌TNF-α,而不诱导ROS的产生。细胞信号传导通路抑制试验结果表明BFP具有体外免疫诱导活性与细胞的NF-κB、JNK MAPK、ERK MAPK和p38 MAPK信号通路的激活相关;F1与细胞的NF-κB、JNK MAPK、ERK MAPK和p38 MAPK信号通路的激活有关;而F2和F3作用机制相似,分别与细胞的NF-κB、JNK MAPK和ERK MAPK信号通路的激活相关。结论:红毛藻多糖具有显著的体外免疫诱导活性,其中分级纯化多糖组分F1和F2是BFP中主要的免疫诱导活性组分;BFP、F1、F2和F3诱导RAW264.7细胞活化释放NO的共同的信号传导通路主要有两条:即NF-κB和JNK MAPK信号途径;而诱导RAW264.7细胞分泌TNF-α的共同信号通路主要是JNK MAPK和ERK MAPK信号途径。

泡叶藻聚糖低分子质量降解片段组成特征及其体外免疫诱导活性

目的:对从泡叶藻(Ascophyllum nodosum)中提取的泡叶藻聚糖进行化学法降解,将得到的低分子质量降解产物进行分离纯化,分析其基本组成特征和体外免疫诱导活性,为泡叶藻聚糖或其他海藻多糖的生物活性和基本结构特征研究提供参考。方法:通过酸水解法和双氧水氧化降解法制备泡叶藻聚糖低分子质量降解片段,利用超滤法和Sephadex G-50凝胶柱层析分离纯化得到4个泡叶藻聚糖低分子质量片段:HCl-F1、HCl-F2、H2O2-F1、H2O2-F2;采用对氨基苯甲酸乙酯柱前衍生样品后进行高效液相色谱及化学方法分析其单糖组成和化学组成;采用高效分子排阻色谱法测定其重均分子质量;通过小鼠巨噬细胞RAW264.7模型分析HCl-F1、HCl-F2、H2O2-F1、H2O2-F2的体外免疫诱导活性。结果:泡叶藻聚糖低分子质量片段的组成特征表明HCl-F1、HCl-F2、H2O2-F1、H2O2-F2均为杂多糖,且单糖组成存在较大差异,其重均分子质量分别为4.80、4.20、5.30、2.30 kDa。免疫活性分析细胞模型结果表明,HCl-F1、HCl-F2、H2O2-F2在所测定质量浓度范围(0~200μg/mL)内能明显诱导RAW264.7细胞活化,释放NO和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),而H2O2-F1诱导RAW264.7细胞释放NO和TNF-α的活性明显低于HCl-F1、HCl-F2和H2O2-F2。结论:泡叶藻聚糖低分子质量降解片段(HCl-F1、HCl-F2和H2O2-F2)具有明显的免疫诱导活性,其中HCl-F1和H2O2-F2的免疫诱导活性明显高于泡叶藻聚糖。结合其化学组成分析的结果,提示泡叶藻聚糖低分子质量降解片段的免疫诱导活性是由单糖组成和硫酸根质量分数共同决定的。

响应面法优化仙茅多糖酶解工艺及体外免疫活性

采用葡聚糖酶对仙茅多糖XMP-1进行酶解,并利用响应面法对酶解工艺进行合理优化。在单因素试验的基础上,采用星点设计模拟葡聚糖酶酶添加量、酶解温度、时间、pH值等4个因素对还原糖生成量综合影响的模型,得到最佳工艺条件,并验证模型的可靠性。通过小鼠巨噬细胞(RAW264.7)分析XMP-11~12 h酶解得到的12批产物的体外免疫活性。结果表明,最佳酶解工艺为酶添加量为56%,温度为52℃,酶解时间为12 h,pH值为6.0,此时还原糖生成量为0.2010 mg/mL。优化结果可靠,与预测值的相对误差小于5%。不同时间段酶解产物对细胞无明显毒害作用,均能诱导RAW264.7细胞活化,释放TNF-α和NO,其中酶解5 h的效果最好。该研究可为仙茅多糖酶降解片段的制备提供理论基础,也可为仙茅多糖在食品领域进一步开发和应用提供科学依据。

Analysis of the Antimicrobial Activity of Antimicrobial Peptides Extracted from Immunized Tenebrio molitor Induced by Different Microbial Treatments Against 26 Pathogens

[Objective] The study was to explore whether antimicrobial activity of the antimicrobial peptides extracted from immunized Tenebrio molitor varied with different pathogens as inducers.[Method]By inducing T.molitor hungry larvaes to generate immune response via feeding with bacteria and with fungi or actinomycete post to pricking,the antimicrobial peptides extracts were obtained by grinding and centrifuging the cultures.Its antimicrobial activity against 26 pathogens was measured by bacteriostatic ring,and evaluated by trisection to four types and inhibitory spectrum.[Result]Both the antimicrobial activity and antimicrobial spectrum of the antimicrobial peptides extracts varied remarkably among different pathogens as inducers.[Conclusion]Bio-control used strains have obvious advantage in inducing the insect to express body fluid immunity material-antimicrobial peptides.

Different morphologic features of rat cochlea progenitor spheres and their implications

Objective: To detect the different morphologic features, developmental regulation, potential of proliferation and differentiation of neonatal rat cochlea progenitor spheres. Methods: We isolated the cochlea sensory epithelium cells from neonatal rats and cultured them in nonadherent conditions to acquire different morphologic spheres. Then we observed the diameter and compositional change of cell colonies in distinct sphere types on day 3, 6, 9 and 12, and summarized the regularity of development and their conversion. We also detected the proliferative activity of distinct spheres by immunohistochemical staining of Abcg2, Nestin and BrdU. After induced spontaneous differentiation, the spheres were detected in the changes of the marker of hair cell, MyosinVIIA; by immunocytochemical staining, we revealed the potential of how different spheres were converted into hair cell-like cells. Results: The acquired three types of suspended spheres are solid, transitional, and hollow. There's morphologic significance among them and they can covert into the other type of spheres among them. The ability of self-renewing and proliferation in distinct spheres vary and all of them have the potential of spontaneously differentiation into hair cell-like cells. Conclusion: All the type of spheres not only has the potential of proliferation and differentiation, but also hasthe potential of spontaneous differentiation into hair cell-like cells. Distinct types of cell spheres neither originate from different progenitor cell subcolonies nor are different stages of the same cell spheres. Solid spheres are most practically useful.