【作者】 王翔岩；

【导师】 齐云；

【作者基本信息】 中国协和医科大学 ， 生药学， 2009， 硕士

【摘要】 目的通过考察肉苁蓉多糖（polysaccharides of Cistanche deserticola CDPS）对小鼠脾淋巴细胞增殖及巨噬细胞活化的作用,研究肉苁蓉多糖的免疫调节药理学活性,并通过不同角度和层次对其吸收特性进行研究。方法MTT法检测小鼠脾淋巴细胞的增殖。环磷酰胺（cyclophosphamide CTX）复制免疫功能低下的动物模型,分别测定正常及免疫低下动物脾脏、胸腺指数。胸腺细胞增殖法测定白细胞介素-2（IL-2）活性。采用中性红法测定小鼠腹腔巨噬细胞吞噬活性;Griess试剂法测定NO释放量;L929细胞法及小鼠胸腺细胞法测定TNF-α及IL-1释放。碳粒廓清法测定小鼠腹腔巨噬细胞吞噬功能。在建立的Caco-2单层细胞模型上通过测定每孔电阻值(R_{true tissue},比较膜两侧碱性磷酸酶（ALP）活性,确定Caco-2细胞单层的可靠性。测定CDPS经Caco-2单层细胞模型A→B 3h转运后受侧浓度,计算其药效P_app值,从体外模型判断CDPS的吸收特性;经体外和体内口服给药药效比较及口服给药途径和腹腔注射给药途径药效的比较从不同角度对肉苁蓉多糖口服吸收特性进行判断。结果CDPS在剂量达7.8mg/L时可促进丝裂原（ConA及LPS）活化淋巴细胞增殖及IL-2的分泌,对未活化淋巴细胞剂量需达25mg/L时加药组与对照组吸光度值才有显著性差异。腹腔给药显示CDPS（125mg/kg）可明显提高正常及免疫低下小鼠的脾指数,对因CTX所致胸腺指数的降低也有显著地对抗作用。CDPS在6.25⁵0mg/L剂量范围内可剂量依赖性地增强BALB/c小鼠腹腔巨噬细胞吞噬中性红能力和促进NO释放;在2.8¹00mg/L剂量范围内可使正常和免疫抑制的RAW264.7细胞NO释放明显增加;在0.56⁷.2mg/L或3.6¹0mg/L范围促进RAW264.7细胞TNF-α或IL-1产生,均具有良好的量效关系。CDPS可在一定程度上提高CTX致免疫功能低下小鼠碳粒廓清指数K。R_{true tissue}值在细胞单层形成过程中稳定增长,细胞模型两侧培养液中ALP活性差异逐步加大,表明建立的Caco-2细胞模型在完整性、细胞极性方面符合药物吸收特性研究的要求,CDPS过Caco-2细胞模型求得P_app值为9.41×10^-8cm/s＜10×10^-6cm/s;对于相同或相关药效指标,CDPS显示出体外及腹腔给药有效,而口服（体内）无效的特征。结论CDPS明显促进小鼠脾淋巴细胞增殖,提高正常及免疫抑制小鼠的脾指数,该作用可能与其促IL-2分泌有关。CDPS可显著提高巨噬细胞吞噬及分泌功能,活化巨噬细胞。显示CDPS是肉苁蓉免疫调节的主要物质基础。但口服吸收不良,因此临床应综合权衡,选择适合的给药方式,以达最佳疗效。更多还原

【Abstract】 Objective To study the immunomodulatory activity of polysaccharides of Cistanche deserticola （CDPS） was assayed by macrophages promoting lymphocyte proliferation and activating macrophage. The absorptive character of CDPS was inveatigated by the comparation of different model systems.Methods The lymphocyte proliferation with or without mitogen was assessed by MTT method in vitro. The immunosuppression mice were induced by cyclophosphamide, and the spleen and thymus were weighted up to determine the immune organ indexes in normal or immunosuppression mice. Thymocytes proliferation was employed to investigate activity of IL-2.The phagocytic ability of peritoneal macrophage （M_Φ） was evaluated with neutral red dye phagocytosis assay; Nitric oxide （NO） production was examined by Griess reaction, the activity of tumor necrosis factor-α（TNF-α） and interleukin-1 （IL-1） secreted were measured by L929 cells bioassay and proliferation of mouse thymocyte. The phagocytic ability of peritoneal macrophage （M_Φ） in vivo was tested with carbon clearance test. Caco-2 cell monolayer was evaluated by determining resistance per well (R_{true tissue}) and comparing bilateral ALP activity in model. The concentration of BL was measured for calculating "pharmacodynamic" P_app values when CDPS transported from A to B across Caco-2 cell monolayer for 3 h, from which the absorption character of CDPS was evaluated. The absorption character of CDPS was assayed from different aspects, which was acrossing Caco-2 monolayer model, comparison of CDPS efficacy in vitro and by oral in vivo, and comparison of CDPS efficacy via oral and i.p. in vivo.Results At the concentration of 7.8 mg/L, CDPS could significantly promote the secretion of IL-2 and proliferation of lymphocyte activated by mitogen （ConA or LPS）, there were significant differences between the absorptance of administration groups and that of control groups when CDPS was up to 25 mg/L in non-activated lymphocyte. CDPS （125 mg/L, i.p.） remarkably increased indexes of spleen in normal or immunosuppression mice, and also improved indexes of immunosuppression mice induced by cyclophosphamide.CDPS at the concentration range of 6.25～50 mg/L enhance the phagocytic ability and NO release of peritoneal macrophage in a dose-dependent manner at the dose of 2.8～100 mg/L significantly increased NO secretion of normal or immunesuppressed RAW264.7 cells. TNF-αproduction by RAW264.7 cell were promoted after incubation of RAW264.7 cells with CDPS at concentration of 0.56～7.2 mg/L. IL-1 release of RAW264.7 cells were obviously elevated by CDPS at dose of 3.6～10 mg/L,and both show good dose-effect relationship. CDPS could enhance the carbon clearance index K to a certain extent in immune- suppressed mice induced by clophosphamide （CTX）.R_{true tissue} and the difference of bilateral ALP activity were increased in cell monolayer developing process, the integrity and polarity of cell monolayer in cell model were satisfactory, which showed that the established Caco-2 cell model can be used to study the intestinal absorption mechanism of oral administration drugs.The P_app values of CDPS was 9.41×10^-8 cm/s, which was less than < 10×10^-6 cm/s; For the same or correlated pharmacodynamics indexes, CDPS showed the character that it was ineffective by oral, but effective in vitro or by ip.Conclusion CDPS can significantly promote the proliferation of splenic lymphocytes, and increased indexes of spleen in normal or immune-suppression mice, which may relate with the promotion of the secretion of IL-2 in splenocyte. CDPS can activate M_Φ, and remarkably promote the phagocytic and secretory of M_Φ. The result suggested that CDPS was the material base of immunomodulatory activity of Cistanche deserticola. But CDPS was malabsorption drug by orial, Therefore the appropriate delivery method should be selected to achieve the best effect in clinical we should weigh the clinical, select the appropriate delivery method to achieve the best effect in clinical practice.更多还原