灵芝S3发酵培养基的优化及其对镰刀菌的抑制

以发酵液对镰刀菌孢子萌发的抑制率为指标,通过单因素试验,研究不同碳源、氮源、生长因子对灵芝S3发酵液抑菌活性的影响。运用响应面法,对各营养组分的含量进行优化。优化后碳源、氮源、生长因子含量分别为：乳糖3.04%,蛋白胨0.28%,VB1 0.0047%,抑菌率为91.71%,比基础发酵培养基增加了42.15%。发酵液作用12h后,光学显微镜下镰刀菌菌丝出现膨大和消融等畸变,并出现典型的“念珠状”形态;透射电镜显示镰刀菌孢子内出现大量空泡、原生质收缩,说明灵芝S3发酵液中的活性物质可有效抑制菌丝生长及孢子萌发。     

灵芝多糖对荷瘤小鼠肿瘤免疫系统的影响

目的:观察灵芝多糖对荷瘤小鼠免疫系统的影响.方法:纯系BALB/c小鼠30只,用U14瘤细胞荷瘤建立动物模型,随机平均分为三组:灵芝多糖治疗组、环磷酰胺治疗组、生理盐水对照组.观察灵芝多糖对荷瘤小鼠NK细胞活性、淋巴细胞转化率、TNF-α等免疫指标的影响.结果:灵芝多糖能够显著提高治疗组小鼠的NK细胞活性、淋巴细胞转化率和血清中TNF-α、IL-2的含量.结论:灵芝多糖能够显著提荷瘤小鼠免疫系统的活性.     

中药提取液对灵芝深层发酵的影响

我国中医药历史悠久 ,中药资源丰富 ,为保证广大人们的身体健康起到巨大作用。但近几十年来 ,我国在中药现代化方面进展缓慢 ,其中原因之一就是中药加工技术陈旧[1] ,近来有学者提出了中药发酵制药技术[1,2 ] 。药用真菌是中药的组成部分 ,它们中的许多种类都能以液体发酵进行生产[3] ,以适当的中药为培养基或在培养基中添加适量的中药 ,利用它们强大的分解转化能力 ,不仅可对中药中的纤维、糖类和蛋白等物质加以利用 ,而且在代谢过程中可能对中药中的一些成分进行转化 ,从而提高中药活性成分在复合制剂中的比例及效价 ;另外中药中的某些成…     

灵芝液体发酵条件的研究

本文研究了某些因素对灵芝液体发酵的影响。4天菌龄的液体菌种发酵效果最好;300毫升三角瓶以装入60毫升培养液发酵较合适;蔗糖、花生饼粉分别为灵芝液体发酵的理想碳、氮源;液体发酵培养基最佳配方为:蔗糖4％、花生饼粉3％、硫酸铵0.15％、磷酸二氢钾0.15％,可得灵芝菌丝体百分干重为0.83,每100毫升发酵液得粗多糖0.3克。     

蜜环菌多糖对小鼠腹腔巨噬细胞免疫功能的影响

从蜜环菌菌索提取的多糖（ＭＨＧ）能在体外显著增强小鼠腹腔巨噬细胞吞噬中性红的作用,并可诱生巨噬细胞产生一氧化氮。在高浓度时,对巨噬细胞分泌ＩＬ－１有一定的作用。     

蛋白质粉对小鼠免疫功能的影响

目的探讨蛋白质粉对正常小鼠免疫调节作用。方法将BALB/c小鼠随机分为3批,每批分为4组,分别进行了小鼠免疫器官/体质量比值测定和小鼠碳廓清实验;绵羊红细胞诱导小鼠DTH、抗体生成细胞检测和血清凝血素测定（HC50）;ConA诱导的小鼠脾淋巴细胞转化实验和乳酸锂脱氢酶法（LDH）测定NK细胞活性;小鼠腹腔巨噬细胞吞噬鸡红细胞实验。结果10.00 g/kg剂量的蛋白质粉可增强绵羊红细胞诱导小鼠DTH能力（P〈0.05）,促进抗体生成细胞数的生成（P〈0.01）。3.33 g/kg和10.00 g/kg剂量组能促进ConA诱导的小鼠脾淋巴细胞转化能力（P〈0.05或P〈0.01）和血清凝血素的生成（P〈0.05）;三个剂量组均能提高小鼠腹腔巨噬细胞吞噬鸡红细胞能力（P〈0.05或P〈0.01）;3.33 g/kg和10.00 g/kg剂量组能提高NK细胞活性（P〈0.05）;但对小鼠碳廓清能力和免疫器官/体重比值无明显影响。结论蛋白质粉对正常小鼠的细胞、体液免疫和单核-巨噬细胞功能和NK功能有促进作用,即具有增强免疫力功能。     

肺炎克雷伯菌生物膜对小鼠巨噬细胞免疫功能的影响

目的研究肺炎克雷伯菌生物膜(BF)对小鼠腹腔巨噬细胞TLRs mRNA和细胞因子表达的影响,探索机体抗BF感染免疫的特点。方法将雄性昆明种小鼠40只随机分成2组,一组腹腔植入体外形成肺炎克雷伯菌BF的硅胶片,建立留置性医疗装置BF感染模型实验组,另一组植入与实验组同等量的浮游菌作为对照组。实时定量PCR分析2组巨噬细胞TLRs mRNA的表达水平,双抗体夹心ELISA法测定细胞因子的含量。结果实验BF组巨噬细胞TLR2、TLR4 mRNA表达量是对照浮游菌组的0.23和0.24倍;而TLR5、TLR9两组表达差异无显著性。实验BF组刺激前后IL-1、IL-2的差值明显低于对照浮游菌组,而IL-4则相反(P0.01)。结论与浮游菌相比,BF能下调小鼠腹腔巨噬细胞TLR2、TLR4的表达,机体的免疫应答朝着Th2型免疫反应发展,这可能是BF相对浮游菌更容易逃脱机体免疫防御系统、引起慢性感染的机制之一。     

参芝发酵产物的抗肿瘤活性及其对小鼠免疫功能的影响

为了探索红参水煎液的灵芝发酵产物对H22荷瘤小鼠的抗肿瘤活性及其对小鼠免疫功能的影响,通过体内抗肿瘤实验和增强免疫功能实验从抑瘤率、对免疫器官的影响指数、对非特异性免疫、体液免疫及细胞免疫的影响5个方面对该产物做了功能性评价。结果表明,在抗肿瘤实验中,参芝发酵产物高剂量组的抑瘤率达到51.65%,脾指数和胸腺指数均高于对照组和环磷酰胺(CTX)组;增强免疫功能实验中,3个实验的给药组小鼠和对照组小鼠相比都有显著性差异(P<0.01)。由此可见,将灵芝与人参在发酵层次上配伍具有显著的抑制荷瘤小鼠肿瘤生长及增强小鼠免疫功能的作用。     

灵芝液体发酵条件的研究

本文采用液体摇瓶培养法,对灵芝（ＧａｎｏｄｅｒｍａＬｕｃｉｄｕｍ）液体发酵的适用温度、摇瓶装量、摇瓶转速、培养基初始ｐＨ、碳、氮源及其最适浓度进行了探讨。结果表明,灵芝液体发酵的适用温度为２５℃,摇瓶装量为１００～１２０ｍｌ／５００ｍｌ三角瓶,摇瓶转速为１２０～１５０ｒｐｍ,培养基初始ｐＨ为４５～５０,适用碳,氮源分别是玉米粉,黄豆饼粉,其最适浓度分别为３％、２５％     

灵芝孢子粉在真菌性肠炎所致腹泻中的应用

目的了解真菌性肠炎所致腹泻的菌种分布特点,探索灵芝孢子粉对其的治疗效果。方法将110例腹泻患者的粪便直接涂片革兰染色镜检。查见较多真菌孢子和菌丝者进行分离鉴定,并采用灵芝孢子粉治疗。结果110例腹泻患者中查见真菌孢子及菌丝者62例;真菌分布白色假丝酵母菌占56．45％,克柔假丝酵母菌占16．13％,热带假丝酵母菌占14．52％,近平滑假丝酵母菌占6．45％,季也蒙假丝酵母菌占6．45％,用灵芝孢子粉治疗真菌性肠炎所致腹泻21d后有效率为96．77％。结论真菌性肠炎所致腹泻以白色假丝酵母菌感染为主,应用灵芝孢子粉治疗方法简单、安全、有效。     

菌草灵芝与段木灵芝的功效成分的比较研究

测定并比较菌草灵芝和不同产地的段木灵芝中的粗多糖、三萜类物质和孢子油中三萜类物质的含量和孢子油得率的差异。结果表明,菌草灵芝和不同产地的段木灵芝中的粗多糖、三萜类物质和孢子油中三萜类物质的含量和孢子油得率存在着差异,菌草灵芝中的粗多糖、三萜类物质和孢子油中三萜类物质的含量和孢子油得率都高于段木灵芝。     

灵芝多糖GLP 抑制小鼠骨肉瘤细胞S-180 在体内生长的作用机制

目的:探讨灵芝多糖成分(GLP)抑制肿瘤的作用机制。方法:在小鼠右腋皮下接种1×106TC-1细胞后7天后,用100mg/kg、200mg/kg和400mg/kg 3种剂量给小鼠口服灌胃给药20天,然后观察肿瘤的重量,并用ELISA检测小鼠血清中IL-2、IL-6和TNF-alpha,用流式细胞仪检测其外周血中CD4+和CD8+。结果:100mg/kg、200mg/kg和400mg/kg 3种剂量给小鼠口服灌胃给药20天,与对照组比较,抑瘤率分别可以达到53%、59%和58%,P<0.05;小鼠外周血血清中的IL-2从1.27ng/mL提高到了2.88ng/mL,P<0.05;TNF-α从1.05ng/mL提高到了1.82ng/mL,P<0.05;而IL-6则没有明显的变化。CD4+细胞水平升高(从54.80%提高到了58.27%),但差异无统计学意义(P>0.05);CD8+细胞明显增多(从24.15%提高到了45.36%),差异有统计学意义(P<0.05)。结论:GLP有明显抑瘤作用,但抑瘤作用与GLP剂量不存在依赖关系。GLP对肿瘤细胞生长的抑制是通过提高小鼠的细胞免疫能力来实现,而并非直接杀伤肿瘤细胞。     

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

 Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34⁺ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34⁺ cells. That the CD34⁺ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34⁺ cells were depleted. The stimulation of CD34⁺ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34⁺ cell levels. Studies also showed that the induction of CD34⁺ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34⁺ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)₂D₃. Received: 8 December 1997 / Accepted: 27 February 1998     

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression

It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.     

Tumor-associated macrophages: Effectors of angiogenesis and tumor progression

Tumor-associated macrophages (TAMs) are a prominent inflammatory cell population in many tumor types residing in both perivascular and avascular, hypoxic regions of these tissues. Analysis of TAMs in human tumor biopsies has shown that they express a variety of tumor-promoting factors and evidence from transgenic murine tumor models has provided unequivocal evidence for the importance of these cells in driving angiogenesis, lymphangiogenesis, immunosuppression, and metastasis. This review will summarize the mechanisms by which monocytes are recruited into tumors, their myriad, tumor-promoting functions within tumors, and the influence of the tumor microenvironment in driving these activities. We also discuss recent attempts to both target/destroy TAMs and exploit them as delivery vehicles for anti-cancer gene therapy.     

Impact of aging on immune modulation by tumor

 Tumor development and aging can each alter immune competence. The present study aimed to determine the impact of Lewis lung carcinoma (LLC) presence on immune parameters of middle-aged (averaging 6.5 months) versus aged (averaging 21.3 months) mice. An age-associated decline in the CD4⁺ cell frequency was seen in freshly isolated spleen and lymph node cells, as well as in cultures stimulated with immobilized anti-CD3. This decline was not further exacerbated by tumor presence. What was prominently inhibited by tumor was the capacity of either splenic or lymph node CD4⁺ cells to become stimulated to express IFN-γ. Spleen and lymph node cultures from aged tumor-bearing mice had the lowest frequency of CD4⁺IFN-γ ⁺ cells and the least amount of secreted IFN-γ. CD8⁺ cells were not affected by aging, but tumor presence reduced the induction of CD8⁺IFN-γ ⁺ cells in lymph node cultures. We previously showed that LLC growth stimulates myelopoiesis, as seen by splenomegaly and the mobilization of immune inhibitory CD34⁺ progenitor cells. Tumor presence in middle-aged mice reduced spleen cell blastogenesis, which was mediated by CD34⁺ cells. Aged mice had reduced blastogenesis, and this was further reduced by presence of tumor. However, neither the age-associated immune dysfunction nor the tumor-induced immune suppression in aged mice was due to CD34⁺ progenitor cells. These studies show how tumor presence can further compromise the immune dysfunction that accompanies aging. In addition, they show that aging impacts on the mechanisms by which tumors inhibit T-cell capabilities, with myelopoiesis-associated CD34⁺ cells mediating the immune depression of middle-aged tumor-bearers and an independent mechanism being responsible for the immune depression in aged tumor-bearing mice. Received: 19 March 2001 / Accepted: 4 May 2001     

灵芝孢子粉水溶性多糖的分离、纯化及结构研究

灵芝孢子粉的热水提取液经醇析,脱脂,去单寡糖后由SepharoseCL 6B柱层析纯化,所得多糖SGL Ⅱ2经高效液相方法鉴定纯度为单一级分,相对分子质量为5 .37×10 4。再经部分酸水解、高碘酸氧化、Smith降解、甲基化分析及IR、GC、GC MS和13 CNMR等方法确定其结构。结果表明多糖SGL Ⅱ 2由葡萄糖和半乳糖组成,为少分支结构,由1→3连接和1→6连接的葡萄糖构成主链,部分1→6连接葡萄糖在3位或4位有分支,侧链为1→4连接的半乳糖,分支末端残基为葡萄糖。     

Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K(2)CO(3) just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C(18) 3.5 micrometer (150x4.6 mm) column (Waters) equipped with a NovaPak C(18) Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 degrees C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min-->(40:60) at 30 min-->(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH(2)PO(4) and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH(2)PO(4) and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r>0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.