灵芝多糖肽对小鼠巨噬细胞自由基的清除作用

目的 :研究灵芝多糖肽对小鼠腹腔巨噬细胞自由基的清除作用。方法 :用四氧嘧啶、叔丁基氢过氧化物 (tBOOH)为氧化剂 ,分别在小鼠体内、体外损伤小鼠腹腔巨噬细胞 ,以DCHF DA为荧光指示剂 ,用共聚焦显微镜观察巨噬细胞的荧光变化 ,并用共聚焦显微镜作时间系列扫描 ,观察巨噬细胞荧光的动态变化。结果 :四氧嘧啶 (75mg·kg-1,iv)、叔丁基氢过氧化物 (7.76× 10 -5mol·L-1)可造成巨噬细胞的氧化损伤 ,使荧光密度增加 ,灵芝多糖肽可减轻其损伤 ,使荧光密度减少。时间系列扫描显示 :随时间改变 ,灵芝多糖肽可减少静息状态下小鼠腹腔巨噬细胞荧光密度 ,也可减少由PMA(5 0nmol·L-1)诱导的呼吸爆发状态下小鼠腹腔巨噬细胞荧光密度。结论 :灵芝多糖肽具有抗氧化作用 ,对小鼠腹腔巨噬细胞自由基有清除作用     

Effects of Ganoderma lucidum polysaccharides on proliferation and cytotoxicity of cytokine-induced killer cells

AIM: To study the effects (and the mechanisms thereof) of Ganoderma lucidum polysaccharides (Gl-PS) on the proliferation and the anti-tumor activity of cytokine-induced killer (CIK) cells, and to make use of CIK cells as a means to investigate the interactions between Gl-PS and cytokines. METHODS: CIK cells were prepared by using the standard protocol as a positive control. Experimental groups also underwent the standard protocol, except that Gl-PS (400 mg/L or 100 mg/L) was added and the dose of anti-CD3 and interleukin-2 they received was reduced by 50% and 75%, respectively. For negative controls, Gl-PS in the experimental protocol was replaced with soluble starch or methylcellulose (400 mg/L or 100 mg/L). CIK cell proliferation, cytotoxicity, and phenotype were determined by using the Trypan blue exclusion method, MTT assay, and flow cytometry. RESULTS: By synergizing cytokines, Gl-PS (400 mg/L or 100 mg/L) could decrease the amount of cytokine in lymphokine activated killer (LAK) cells and CIK cells culture, but had no significant effect on the proliferation, cytotoxicity, or phenotype of LAK cells, or CIK cells induced by cytokines at higher doses alone, in which CIK cells expanded about 80-fold and the main effectors, CD3+NK1.1+ cells, expanded by more than 15%. The cytotoxicity of CIK cells in experimental groups was 79.3%+/-4.7%, 76.9%+/-6.8% versus the positive control 80.7%+/-6.8% against P815 (P>0.05) and 88.9%+/-5.5%, 84.7%+/-7.9% versus the positive control 89.8%+/-4.5% against YAC-1 (P>0.05). The activity of Gl-PS could mostly be blocked by anti-CR3. CONCLUSION: Gl-PS was shown to be a promising biological response modifier and immune potentiator. The effect of Gl-PS on CIK cells is possibly mediated primarily through complement receptor type 3.     

灵芝多糖肽对氧自由基损伤巨噬细胞的保护作用

目的:研究灵芝多糖肽(GLPP)在离体和整体水平对氧自由基(ROS)(tBOOH为氧化剂)损伤巨噬细胞的保护作用.方法:以tBOOH为氧化剂损伤小鼠腹腔巨噬细胞,以MTT法分析小鼠巨噬细胞存活率,在光镜和电子显微镜下观察细胞的形态改变.结果:GLPP50,100,200 mg/kg腹腔注射5天,能抑制巨噬细胞膜样变性和坏死,细胞存活率提高.在培养的巨噬细胞中加入 GLPP 3.125,12.5,50,200 mg/L,产生相似的保护作用.电镜观察发现,GLPP(100mg/kg)腹腔注射5天可保护细胞器如线粒体免受tBOOH的损伤.结论:GLPP有显著的清除氧自由基和抗氧化作用.     

灵芝多糖肽对自由基所致的腹腔巨噬细胞早期损伤的影响

目的　以膜电位方法进一步研究灵芝多糖肽(GLPP)对自由基损伤的巨噬细胞线粒体的影响。方法　以叔丁基氢过氧化物(tBOOH)为氧化剂,建立小鼠巨噬细胞体外氧化损伤模型,以罗丹明1 2 3(Rh1 2 3 )为荧光染色剂,以激光共聚焦显微镜检测细胞线粒体膜电位改变。结果　tBOOH氧化剂可损伤线粒体膜,使线粒体膜电位降低,GLPP体内( 1 0 0mg·kg- 1 ig 5d)及体外给药(GLPP 1 0mg·L- 1)均可对抗tBOOH自由基损伤,可使因自由基损伤而降低的巨噬细胞线粒体膜电位恢复。结论　GLPP体内体外给药可减轻自由基损伤,使损伤的线粒体膜电位恢复     

灵芝多糖对小鼠腹腔巨噬细胞活性氧自由基的影响

为进一步探讨灵芝多糖 ( GLP)免疫调节作用机理 ,采用激光扫描共聚焦显微镜技术 ,动态监测灵芝多糖均一体 GLB7对小鼠腹腔巨噬细胞 ( M )活性氧自由基含量的影响 .以 GLB7( 2 0 mg· L-1)刺激 M ,发现探针 DCHF- DA的荧光强度明显下降 ,与静息水平相比 ,平均下降 ( 36± 6) % ( n=6,P<0 .0 5) ;同时还能拮抗 PMA引起的 M 呼吸爆发 ,荧光强度下降幅度为 ( 1 9± 4) % ( n=6,P<0 .0 5) .结果证明 :GLB7能减少 M 内活性氧自由基的生成 ,具有清除活性氧的作用 ,这也是 GLB7产生免疫增强和抗衰老作用的重要途径 .     

Hypoglycemic effect of Ganoderma lucidum polysaccharides

AIM: To investigate the hypoglycemic effect of Ganoderma lucidum polysaccharides (Gl-PS) in the normal fasted mice and its possible mechanism. METHODS: Normal fasted mice were given a single dose of Gl-PS 25, 50, and 100mg/kg by ip and the serum glucose was measured at 0, 3, and 6h after administration. Gl-PS 100mg/kg were also given by ip and the serum glucose and insulin levels were measured at 0min, 30min, 1h, 3h, 6h, and 12h Pancreatic islets were isolated and incubated with glucose 5.6mmol/L and different concentration of Gl-PS, the insulin content of islets and insulin release were examined. The islets fluorescent intensity of [Ca^2 ]i was also studied with a confocal microscope. Verapamil and egtazic acid were used to testify whether the insulin-releasing effect of Gl-PS was mediated by its ability to raise the Ca^2 influx. RESULTS: Gl-PS dose-dependently lowered the serum glucose levels at 3h and 6h after administration. Gl-PS 100mg/kg raised the circulating insulin levels at 1h after administration. In vitro, Gl-PS had no effect on islets insulin content, but it stimulated the insulin release after incubation with glucose 5.6mmol/L. Confocal microscope showed that Gl-PS 100mg/L had the capacity toraise the [Ca^2 ]i. The insulin-releasing effect of Gl-PS was inhibited by verapamil/egtazic acid. CONCLUSION:Gl-PS possesses the hypoglycemic effect on normal mice; one mechanism is through its insulin-releasing activity due to a facilitation of Ca^2 inflow to the pancreatic β cells.     

段木栽培及袋栽灵芝多糖对体外培养小鼠脾淋巴细胞增殖活性的比较

目的 比较段木栽培灵芝多糖(wood-cultured Ganoderma lucidum polysaccharides, GL-PS-WC) 及袋栽灵芝多糖(bag-cultured Ganoderma lucidum polysaccharides, GL-PS-BC)对体外培养小鼠脾淋巴细胞增殖活性的影响,探讨袋栽灵芝多糖替代段木栽培灵芝多糖的可能性。 方法检测两种灵芝多糖对混合淋巴细胞培养(MLC)反应的影响;观察对刀豆蛋白A (Con A)、细菌脂多糖(LPS)诱导淋巴细胞增殖的影响以及对环孢素A (CsA)、丝裂霉素C(Mit C)、足叶乙苷(VP-16) 等抑制MLC反应的影响。结果当质量浓度为0.2～12.8 mg·L^-1时,两种灵芝多糖均可促进MLC反应,增强Con A或LPS诱导的淋巴细胞增殖,并拮抗CsA, Mit C或VP-16对MLC反应的抑制作用。未发现两种多糖之间有显著性差异。结论GL-PS-WC及GL-PS-BC对体外培养脾淋巴细胞的增殖活性有类似作用。     

灵芝孢子多糖两纯组分的单糖组成研究

目的：对灵芝孢子多糖的2个纯多糖组分GLPSl和GLPS3的单糖组成进行分析。方法：将灵芝孢子多糖原料精制，SephacrylS一400SF型凝胶分离纯化得2个主要组分GLPSl和GLPS3，经酸水解后TLC法和HPLC法鉴别单糖组成，改良的1一苯基一3一甲基一5一吡唑啉酮（PMP）柱前衍生化HPLC法分析单糖组成比例，同时采用LC—MS联用技术鉴定单糖PMP衍生化产物。结果：GLPSl的单糖组成为D一甘露糖：D一葡萄糖：D一半乳糖=1：9．228：1．474；GLPS3的单糖组成为D一甘露糖：D．葡萄糖：D．半乳糖=1：15．900：3．686。结论：灵芝孢子多糖二纯组分均含D一甘露糖，D．葡萄糖和D一半乳糖，仅各单糖的比例不同。     

灵芝多糖对体外树突状细胞诱导细胞毒性T-淋巴细胞的调节作用(英文)

目的:研究灵芝多糖(Gl-PS)在抗原提呈阶段对体外培养树突状细胞(DC)诱导特异性细胞毒性T-淋巴细胞(CTL)功能的调节及其机制。方法:体外培养小鼠骨髓来源DC经P815肿瘤细胞冻融抗原冲击致敏,并与不同浓度Gl-PS(0.8,3.2,或12.8 mg/L)共培养。成熟DC与脾淋巴细胞共培养诱导P815特异性CTL生成。第5天收集各组悬浮细胞及培养上清,采用乳酸脱氢酶法比较CTL特异性杀伤活性;RT-PCR法测定T扰素γ(IFNγ)及颗粒酶B mRNA在CTL的表达;ELISA或Western blot法检测IFNγ或颗粒酶B蛋白的表达。结果:3种浓度的Gl-PS均可增高培养上清中释放的LDH活性(P<0.01);增加CTL表达IFN7及颗粒酶B mRNA(IFNγ:Gl-PS 12.8 mg/L组与RPMI-1640组,P<0.05;颗粒酶B:P<0.01);促进CTL培养上清中IFNγ蛋白生成(P<0.05)以及CTL表达颗粒酶B蛋白(Gl-PS 12.8 mg/L组与RPMI-1640组,P<0.05)。结论:Gl-PS可在抗原提呈阶段促进P815肿瘤冻融抗原冲击致敏DC所诱导的特异性CTL的杀伤活性,其机制可能是通过IFNγ及颗粒酶B途径的调节。     

灵芝多糖对糖尿病大鼠心肌纤维化的保护作用

目的:研究灵芝多糖对2型糖尿病大鼠心肌纤维化的保护作用。方法:SD大鼠高脂饮食4周后,注射STZ(30 mg.kg-1)复制2型糖尿病大鼠模型,造模成功后,将大鼠分为正常对照组、糖尿病组、灵芝多糖低、中、高剂量组(分别灌胃给予灵芝多糖200,400,800 mg.kg-1)及小檗碱对照组(灌胃小檗碱30 mg.kg-1)。治疗8周后,测量大鼠空腹血糖,VG染色观察心肌纤维化程度,荧光分光光度计法检测大鼠血清晚期糖基化终末产物(AGE)的含量,碱水解法测定心肌羟脯氨酸含量,胃蛋白酶限制性降解法测定胶原交联程度,并测量心肌和血清中过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)等氧化应激指标。结果:中高剂量灵芝多糖在降低2型糖尿病大鼠血糖水平的同时,可使VG染色血管周围和间质胶原明显减少,心肌组织中羟脯氨酸含量下降,心肌交联程度得到明显改善,血清中AGE含量降低,心肌和血清中CAT和GSH-PX活力得到显著增强。结论:灵芝多糖可降低2型糖尿病大鼠心肌胶原纤维总量和交联程度,其机制可能与灵芝多糖降低血清AGE含量,增强机体内CAT和GSH-PX活性有关。     

灵芝多糖对肿瘤细胞与内皮细胞相互作用的影响

目的研究灵芝多糖(GlPS)抗肿瘤作用及其机制。方法通过相差显微镜和间接免疫荧光的方法鉴定了人脐静脉内皮细胞(HUVECs)。采用MTT法检测GlPS对人前列腺癌细胞(PC-3M)和HUVECs增殖的影响。检测GlPS对PC-3M细胞与HUVECs粘附的影响。采用transwell双层小室观察GlPS对PC-3M迁移穿过单层HUVECs的影响。结果GlPS对PC-3M无直接细胞毒作用,对HUVECs增殖无显著性作用。GlPS能够减少粘附和迁移穿过单层内皮细胞的肿瘤细胞数目。结论灵芝多糖通过抑制肿瘤细胞粘附并迁移穿过内皮细胞发挥其抗肿瘤作用。     

灵芝多糖肽对小鼠腹腔巨噬细胞一氧化氮产生的影响

目的　研究灵芝多糖肽 (GLPP)对小鼠腹腔巨噬细胞一氧化氮产生的影响并探讨其作用机制。方法　以Griess法 ,观察GLPP对LPS诱导小鼠腹腔巨噬细胞一氧化氮(NO)产生的影响 ;以免疫组化法检测诱导型一氧化氮合成酶 (iNOS)的表达 ,观察GLPP对iNOS的影响。结果　GLPP(2 5～ 2 0 0mg·kg-1)灌胃给药 5d或体外给药 (3 12 5～ 2 0 0mg·L-1)均可促进巨噬细胞NO释放 ,但对LPS刺激NO的释放影响不大 ;GLPP(10 0mg·kg-1)灌胃给药 5d或体外给药 (10mg·L-1)均可使巨噬细胞iNOS含量增加。结论　GLPP可增加小鼠腹腔巨噬细胞NO产生 ,其机制可能与其促进巨噬细胞iNOS合成有关。     

Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells

Objectives Tumour cells produce factors such as interleukin 10 (IL‐10), transforming growth factor β1 (TGF‐β1) and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. One of the most important goals of tumour immunotherapy is to antagonize this suppression on immune cells. Ganoderma lucidum polysaccharides (Gl‐PS) may have this potential. The purpose of this study was to determine the antagonistic effects of Gl‐PS on the suppression induced by B16F10 melanoma cell culture supernatant (B16F10‐CS) on lymphocytes. Methods Gl‐PS was used on lymphocytes incubated with B16F10‐CS. Enzyme‐linked immunosorbent assay was used to determine the levels of IL‐10, TGF‐β1 and VEGF in B16F10‐CS. The MTT assay was used to determine the proliferation of lymphocytes. Immunocytochemistry and Western blot assay were used to determine perforin and granzyme B production in lymphocytes. Key findings There were elevated levels of IL‐10, TGF‐β1 and VEGF in B16F10‐CS. The lymphocyte proliferation, and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction, were suppressed by B16F10‐CS. This suppression was fully or partially antagonized by Gl‐PS. Conclusions B16F10‐CS suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction. This suppression may be associated with elevated levels of immunosuppressive IL‐10, TGF‐β1 and VEGF in B16F10‐CS. Gl‐PS had antagonistic effects on the immunosuppression induced by B16F10‐CS, suggesting the potential for Gl‐PS in cancer immunotherapy.     

灵芝多糖对大鼠胰岛细胞分泌胰岛素功能的影响

目的 :探讨灵芝多糖 (Gl PS)对胰岛细胞分泌胰岛素的影响。方法 :分离大鼠胰岛细胞 ,分别观察在葡萄糖为 5 .6和 1 6.7mmol·L- 1 时 ,Gl PS对胰岛细胞分泌胰岛素的影响。进一步加入不同的抑制剂观察Gl PS对胰岛细胞分泌胰岛素的影响。采用Westernblotting观察Gl PS对胰岛细胞葡萄糖转运蛋白 2 (GLUT2 )表达的影响。结果 :在 5 .6mmol·L- 1葡萄糖时 ,1 0 0mg·L- 1 的Gl PS可明显促进胰岛细胞胰岛素的分泌 ;当葡萄糖浓度为 1 6.7mmol·L- 1 时 ,5 0和 1 0 0mg·L- 1 的Gl PS均可明显促进胰岛细胞胰岛素的分泌。维拉帕米或维拉帕米 +EGTA均可显著抑制 5 .6和 1 6.7mmol·L- 1 葡萄糖时胰岛素的分泌 ,维拉帕米只能部分抑制Gl PS的促胰岛素分泌作用 ,而维拉帕米 +EGTA则可完全抑制Gl PS的这种作用。在葡萄糖浓度为 5 .6和 1 6.7mmol·L- 1 时 ,Gl PS 5 0和 1 0 0mg·L- 1 均能明显促进胰岛细胞GLUT2的蛋白表达。结论 :Gl PS可能通过促进胰岛细胞GLUT2蛋白的表达从而有助于葡萄糖转运入B细胞 ,促进葡萄糖的代谢 ,引起胰岛细胞外Ca2 + 内流而起到促胰岛素释放的作用     

Effect of polysaccharides from Ganoderma lucidum on streptozotocin-induced diabetic nephropathy in mice

The effects of Ganoderma lucidum polysaccharides (GL-PS) on renal complication in streptozotocin-induced diabetic mice have been investigated in the present study. C57BL/6J mice were made diabetic by injection of streptozotocin and GL-PS (125 and 250 mg kg^- 1) was administered for 8 weeks. Body weight was monitored every week. Serum glucose, creatinine (Cr), blood urea nitrogen (BUN), triglyceride (TG) and urinary albumin excretion (UAE) were measured after 8 weeks of treatment. Glomerular size and mesangial matrix index were assayed by morphometric analysis. Renal expression of transforming growth factor-β₁ (TGF-β₁) were determined by immunochemistry. Renal malondialdehyde (MDA) level and superoxide dismutase (SOD) activities were also evaluated. GL-PS was able to reduce the serum Cr and BUN levels and UAE compared with diabetic model mice in a dose-dependent manner. Increasing serum glucose and triglyceride levels in diabetic mice could also be lowered by GL-PS. Moreover, GL-PS had the capacity to improve the renal morphometric changes and oxidative stress state of diabetic mice. In summary, GL-PS can improve the metabolic abnormalities of diabetic mice and prevent or delay the progression of diabetic renal complications.     

Effect of polysaccharides from Ganoderma lucidum on streptozotocin-induced diabetic nephropathy in mice

The effects of Ganoderma lucidum polysaccharides (GL-PS) on renal complication in streptozotocin-induced diabetic mice have been investigated in the present study. C57BL/6J mice were made diabetic by injection of streptozotocin and GL-PS (125 and 250 mg kg^? 1) was administered for 8 weeks. Body weight was monitored every week. Serum glucose, creatinine (Cr), blood urea nitrogen (BUN), triglyceride (TG) and urinary albumin excretion (UAE) were measured after 8 weeks of treatment. Glomerular size and mesangial matrix index were assayed by morphometric analysis. Renal expression of transforming growth factor-β₁ (TGF-β₁) were determined by immunochemistry. Renal malondialdehyde (MDA) level and superoxide dismutase (SOD) activities were also evaluated. GL-PS was able to reduce the serum Cr and BUN levels and UAE compared with diabetic model mice in a dose-dependent manner. Increasing serum glucose and triglyceride levels in diabetic mice could also be lowered by GL-PS. Moreover, GL-PS had the capacity to improve the renal morphometric changes and oxidative stress state of diabetic mice. In summary, GL-PS can improve the metabolic abnormalities of diabetic mice and prevent or delay the progression of diabetic renal complications.     

Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms

Ganoderma lucidum (G lucidum) is a medicinal fungus with a variety of biological activities. It has long been used as a folk remedy for promotion of health and longevity in China and other oriental countries. The most attractive character of this kind of medicinal fungus is its immunomodulatory and anti-tumor activities. Large numbers of studies have shown that G lucidum modulate many components of the immune system such as the antigen-presenting cells, NK cells, T and B lymphocytes, The water extract and the polysaccharides fraction of G lucidum exhibited significant anti-tumor effect in several tumor-bearing animals mainly through its immunoenhancing activity. Recent studies also showed that the alcohol extract or the triterpene fraction of G lucidum possessed anti-tumor effect, which seemed to be related to the cytotoxic activity against tumor cells directly. Preliminary study indicated that antiangiogenic effect may be involved antitumor activity of G lucidum.     

灵芝孢子粉与子实体中三萜类化合物、多糖及重金属元素的含量比较

目的:比较灵芝孢子粉与子实体中三萜类化合物、多糖及重金属元素的含量差异。方法:采用高效液相色谱(HPLC)法分析灵芝孢子粉和子实体样品中的三萜类化合物,用电感耦合等离子体质谱(ICP-MS)技术对上述样品中5种重金属元素的含量进行测定,用蒽酮-硫酸法测定其中多糖的含量。结果:灵芝孢子粉和子实体中三萜类化合物的HPLC特征图谱差异明显;灵芝孢子粉中多糖的含量高于子实体中的含量;而重金属元素的含量二者没有显著差异。结论:本试验结果可为灵芝孢子粉与子实体药理作用的差异提供理论依据。     

赤芝子实体的化学成分及其抗肿瘤活性

目的:研究赤芝子实体的化学成分及其体外抗肿瘤活性。方法:用硅胶柱色谱法,Sephadex LH-20柱色谱法及制备型高效液相色谱法进行分离纯化,依据理化性质和光谱数据鉴定化合物结构;用MTT法测试化合物体外抗肿瘤活性。结果:分离得到6个化合物,其结构鉴定为ganoderone A(1)、麦角甾醇(2)、麦角甾醇过氧化物(3)、麦角甾-7、22-二烯-3β,5α,6β-三醇(4)、灵芝酸DM (5)和7-oxo-灵芝酸Z(6);化合物1,3对人慢性粒细胞白血病细胞K562和人结肠癌细胞SW620的IC₅₀分别为9.61,10.70 mg·L^-1和22.38,32.48 mg·L^-1,化合物1~6对人急性髓系白血病细胞HL60的IC₅₀分别为7.14,11.07,2.80,7.54,51.82和7.39 mg·L^-1。结论:ganoderone A为首次从赤芝中分离得到;灵芝中的甾醇及三萜类化合物均有一定抗肿瘤活性。     

赤芝多糖肽GL-PP-3A的分离纯化和结构研究

分离纯化赤芝多糖肽，得到具有活性部位GL-PP-3A，对其进行结构分析。水提醇沉透析得赤芝多糖肽，经分级沉淀，Bio-Gel P-10柱色谱纯化得GL-PP-3A。经HPGPC、糖基组成、甲基化分析、 ¹H NMR和 ¹³C NMR等方法研究分析GL-PP-3A的结构。GL-PP-3A是以葡萄糖为主的杂多糖，含Rha、Xyl、Man、Gal、Glc糖残基和17种氨基酸。其M_w为1.7×10⁴；M_n为1.1×10⁴；M_w/M_n为1.49。M_p为1.3×10⁴。该多糖主链由1,6-或1,3-连接的β-D-Glcp构成，二者的比例约为2∶1，在部分1,6-连接的主链葡萄糖的2或3位有分支，分支的非还原末端均主要为β-D-Glcp,少量为鼠李糖，分支内部含有1～3个1,6-连接的β-D-Galp或1,3-连接的α-D-Manp。