硫酸酯化马尾松花粉多糖对小鼠脾脏B淋巴细胞免疫调节作用的研究  

Infl uences of Sulfated Polysaccharide from Pine(Pinus massoniana) Pollen on the Immunomodulatory Effects of B Lymphocytes in Mice

作　　者：刘铭[1] 李娜娜[1] 耿越[1] 

机构地区：[1]山东省动物抗性生物学重点实验室,山东师范大学生命科学学院,济南250014

出　　处：《中国细胞生物学学报》2014年第4期461-469,共9页Chinese Journal of Cell Biology

基　　金：山东省自然科学基金(批准号:Y2008D13)资助的课题~~

摘　　要：研究60%乙醇提取的马尾松花粉多糖组分D(PPM60-D)及其硫酸酯化物(SPPM60-D)对小鼠脾脏B淋巴细胞增殖、细胞内游离钙离子浓度([Ca2+]i)及抗体生成的影响。水煮醇沉法提取得到粗多糖,乙醇分级沉淀得到60%乙醇沉淀多糖PPM60,Sephacryl S-400HR分离纯化得到多糖组分D,用氯磺酸–吡啶法对组分D进行硫酸酯化,尼龙毛法分离B淋巴细胞,MTT法测定其增殖,荧光分光光度计测定B淋巴细胞[Ca2+]i,溶血空斑实验(PFC)和定量溶血分光光度(QHS)法测定B细胞抗体生成情况。结果显示,SPPM60-D相对于PPM60-D能更显著地提高B淋巴细胞的增殖以及[Ca2+]i(P<0.01);经TAK-242、LY294002、U73122、低分子肝素、维拉帕米和2-APB抑制剂作用后,均可抑制SPPM60-D和PPM60-D所致的[Ca2+]i升高(P<0.05或P<0.01);PFC和QHS检测证实,SPPM60-D对于促进B淋巴细胞的分化及抗体的生成有显著作用,而PPM60-D的作用较弱。以上研究表明,PPM60-D经过硫酸酯化改性后,活性明显提高,推测SPPM60-D可与B淋巴细胞上TOLL样受体4(TLR4)结合,通过TLR4-PI3K-PLC-IP3R信号通路使钙库释放激活的钙通道(CRAC)打开,从而使[Ca2+]i升高来激活B淋巴细胞,进而提高其体外增殖和抗体生成能力。We investigated the influences of a polysaccharide component D （PPM60-D） and its sulphate （SPPM60-D） from masson pine pollen on the proliferation, intracellular free calcium concentration （[Ca2＋]i） and antibody production of B lymphocytes in mice. Polysaccharides （PPM60） were extracted from masson pine pollen with hot water and precipitated by 60% ethanol. PPM60-D was purified from PPM60 with Sephacryl S-400HR. Sulfated polysaccharides （SPPM60-D） was derivated by chlorosulfonic acid-pyridine method. B lymphocytes were separated through nylon wool column. [Ca2＋]i） of B lymphocytes were measured by fluorescence sepectrophotom- eter. The antibody production were measured by PFC and QHS methods. The results showed that SPPM60-D could more significantly improve the proliferation, [Ca2＋]i and antibody production of B lymphocytes than PPM60-D （P〈0.01）. TAK-242, LY294002, U73122, low molecular heparin, verapamil and 2-APB could inhibit the increase- ment of [Ca2＋]i in B lymphocytes which was induced by PPM60-D and SPPM60-D （P〈0.05 or P〈0.01）. These sug- gested that SPPM60-D had higher activity than PPM60-D and one of the possible receptors of SPPM60-D was toll like receptor 4 （TLR4）. We speculated that SPPM60-D could increase [Ca2＋]i via Ca2＋ release-activated Ca2＋ （CRAC） channel which was activated by TLR4-PI3K-PLC-IP3R signaling pathway, so that it could improve the ability of proliferation and antibody production of B lymphocytes.

关 键 词：马尾松花粉 硫酸酯化多糖 B淋巴细胞 增殖 [Ca2+]i) 抗体生成 

分 类 号：R392[医药卫生—免疫学]