灵芝菌丝体多糖对K562细胞的抑制作用及免疫调节作用研究

目的研究灵芝菌丝体多糖（ganoderma lucidum mycelia polysaccharides,GLMP）对K562白血病细胞及小鼠体外脾淋巴细胞增殖的影响。方法 MTT法检测不同浓度GLMP对K562白血病细胞增殖及对小鼠体外脾淋巴细胞增殖的作用;血细胞计数方法绘制生长曲线。结果不同浓度的GLMP处理K562细胞48h后,50mg/L及以上浓度抑制率与对照组比较差异有统计学意义（P〈0.01）,而且随浓度的增加抑制率增加。作用48h的IC50为270.2mg/L。生长曲线也表明随时间的延长和浓度的增加,细胞增殖能力下降。GLMP作用小鼠体外脾淋巴细胞48h后,在多糖浓度为50、100mg/L和25、50、100mg/L时能显著刺激静止性和激活型细胞的增殖。结论 GLMP可以抑制K562白血病细胞增殖而且具有免疫调节能力。     

二甲基甲酰胺对小鼠体液免疫的影响

本文采用Simpson定量溶血分光光度法检测了DMF对小鼠初次体液免疫及二次体液免疫反应的影响,同时以同位素掺入法研究TDMF在体外对LPS诱导的脾淋巴细胞增殖的作用。结果显示,以27～432mg/kg DMF给昆明种小鼠皮下注射,可引起初次体液免疫的明显增强,对二次体液免疫呈低剂量增强、高剂量抑制的双相反应。体外试验表明,在10～15m molDMF存在下,LPS诱导的C_(57)BL/6J小鼠脾淋巴细胞增殖受到明显抑制,提示小鼠体液免疫受到一定的影响。     

儿茶素对环磷酰胺诱导小鼠迟发性超敏反应的影响*

目的:研究儿茶素对环磷酰胺诱导小鼠迟发性超敏反应的影响。方法:以2,4-二硝基氯苯(DNCB)诱导小鼠迟发性超敏反应(DTH),致敏后立即腹腔注射(ip)环磷酰胺(Cy)125 mg.kg-1和致敏前3 d ip Cy 250mg.kg-1分别诱导小鼠降低和增强的DTH反应,用耳肿胀度、胸腺指数和脾指数作为主要检测指标。以ConA诱导胸腺T淋巴细胞,观察不同剂量或浓度的儿茶素体内外对胸腺T淋巴细胞增殖和IL-2生成的影响。MTT法检测胸腺T淋巴细胞增殖,ConA诱导的胸腺T淋巴细胞培养上清中IL-2活性检测采用活化的小鼠胸腺细胞MTT比色法。结果:于致敏前3 d灌胃儿茶素30,60,120 mg.kg-1.d-1,连续7 d,在Cy诱导的DTH反应降低小鼠模型,儿茶素在60,120 mg.kg-1剂量可显著上调其低下的耳肿胀度、胸腺和脾指数,并上调胸腺T淋巴细胞增殖反应以及产生的IL-2水平;在Cy诱导的DTH反应增强小鼠模型,儿茶素60,120 mg.kg-1可显著下调其增高的耳肿胀、胸腺和脾指数,儿茶素30,60,120 mg.kg-1可显著抑制其增强的胸腺T淋巴细胞增殖反应以及产生的IL-2水平。儿茶素体外在25~200 mg.L-1浓度时能增强Cy诱导的DTH反应降低的小鼠胸腺T淋巴细胞增殖和IL-2水平;儿茶素在12.5~200 mg.L-1浓度时能降低Cy诱导的DTH反应增强的小鼠胸腺T淋巴细胞增殖和IL-2水平。结论:儿茶素对小鼠的细胞免疫具有调节作用。     

白细胞介素Ⅰ的检测及白芍总甙对其产生的影响

本文对小鼠胸腺细胞增殖法检测IL—1的条件及白芍总甙(TGP)对其产生的影响进行了探讨。结果表明,LPS诱导大鼠腹腔Mφ产生IL—1的最佳浓度为8mg/L,诱导出富含IL—1的上清经活化的小鼠脾细胞检测表明无明显IL—2污染。富含IL—1的待测上清协同亚适量Con A诱导小鼠胸腺细胞增殖反应的最佳稀释度为1:90。TGP和亚适浓度LPS与大鼠腹腔Mφ体外共育6h后洗去药物和LPS,结果表明,TGP(0.5～12.5mg/L)可浓度依赖性地增加IL—1的产生,但高浓度TGP(62.5～125mg/L)时,IL—1产生显著降低,量效曲线呈钟罩形趋势,提示TGP对IL—1的产生具有双向作用。     

螺旋藻多糖硫酸酯化修饰前后抗肿瘤及免疫活性的研究

比较螺旋藻多糖硫酸酯化修饰前后体内外抗肿瘤及免疫活性.采用MTT比色法研究药物在体外对人肿瘤细胞株的抑制作用,促进正常小鼠脾淋巴细胞的增殖活性以及对荷瘤小鼠NK和CTL细胞活性的影响.采用移植性肿瘤实验方法考察了药物对小鼠S180肉瘤的抑制作用.结果显示,螺旋藻多糖（NPSP）对肿瘤细胞株几乎无细胞毒作用,硫酸酯化螺旋藻多糖（SNPSP）对肿瘤细胞株具有显著的细胞毒作用,其中对SMMC-7721人肝癌细胞株抑制率最高达50%.NPSP50 mg/kg对小鼠S180肉瘤无抑制,相同剂量的SNPSP对小鼠S180肉瘤的抑制率达35.42%.NPSP具有促进脾淋巴细胞增殖作用,但对ConA和LPS诱导的脾淋巴细胞增殖反应无促进作用,SNPSP促进脾淋巴细胞增殖作用较NPSP增强,同时对ConA和LPS诱导的脾淋巴细胞增殖反应也具有明显的促进作用.NPSP和SNPSP均能促进荷瘤小鼠NK细胞和CTL细胞活性,其中SNPSP促进CTL细胞活性较NPSP增强.     

阿糖胞苷引起毛囊损伤离体模型的建立以及他克莫司对其的逆转作用

目的建立阿糖胞苷(Ara-c)诱导毛囊损伤的离体器官培养模型,并观察他克莫司(FK506)对Ara-c诱导的小鼠触须毛囊损伤的逆转作用.方法用离体器官培养的方法在倒置显微镜底下每日测量空白组、FK506 Ara-c和单纯Ara-c作用下毛囊生长长度、记录生长天数,以及液相闪烁仪测量同位素^3H-TdR的掺入率.结果 Ara-c10mg/L和25mg/L的浓度,作用2.5h能明显抑制毛囊生长和DNA合成,缩短毛囊在体外的生长时间,抑制毛囊球部细胞增殖.0.01～0.3mg/L的FK506能改善10mg/L Ara-c引起的毛囊生长和DNA合成的抑制,体外生长时间缩短以及毛囊球部细胞增殖抑制.0.1mg/L的FK506对25mg/L Ara-c引起的毛囊损伤也有相似的改善作用.结论 Ara-c诱导毛囊损伤的离体模型可以用于研究化疗脱发的机制和某些因子及药物对它的干预作用,FK506在体外对Ara-c诱导的毛囊损伤有修复作用,是治疗化疗后脱发的潜在药物.     

香菇多糖(KS-2)刺激小鼠免疫功能的实验研究

香菇多糖(KS—2)腹腔注射能明显增强小鼠脾抗体分泌细胞数(PFC),最佳给药剂量为100mg/kg,也可明显增强小鼠对BSA诱导的迟发型超敏反应性(DTH)。它在体外对ConA,LPS引起的小鼠淋巴细胞增殖反应无影响。     

阿糖胞苷引起毛囊损伤离体模型的建立以及他克莫司对其的逆转作用

目的　建立阿糖胞苷(Ara-c)诱导毛囊损伤的离体器官培养模型,并观察他克莫司(FK506)对Ara -c诱导的小鼠触须毛囊损伤的逆转作用。方法　用离体器官培养的方法在倒置显微镜底下每日测量空白组、FK506+Ara -c和单纯Ara c作用下毛囊生长长度、记录生长天数,以及液相闪烁仪测量同位素³H-TdR的掺入率。结果　Ara-c10mg/L和25mg/L的浓度,作用2. 5h能明显抑制毛囊生长和DNA合成,缩短毛囊在体外的生长时间,抑制毛囊球部细胞增殖。0.01 ～0. 3mg/L的FK506能改善10mg/Lara -c引起的毛囊生长和DNA合成的抑制,体外生长时间缩短以及毛囊球部细胞增殖抑制。0. 1mg/L的FK506对25mg/Lara -c引起的毛囊损伤也有相似的改善作用。结论　Ara- c诱导毛囊损伤的离体模型可以用于研究化疗脱发的机制和某些因子及药物对它的干预作用,FK506在体外对Ara- c诱导的毛囊损伤有修复作用,是治疗化疗后脱发的潜在药物。     

褪黑素对不同月龄小鼠免疫反应的影响及其机制

目的:研究褪黑素对不同月龄小鼠免疫反应的影响及其作用机制。方法:淋巴细胞增殖采用MTT法;IL-2活性检测 采用激活小鼠脾淋巴细胞增殖法;cAMP测定用竞争蛋白结合法;甲硫氨酸脑啡肽的测定采用放免法。结果:6月龄和11月龄BALB/c小鼠胸腺淋巴细胞增殖能力和IL-2的产生均降低,褪黑素(5mg/kg或30mg/kg,ig×7d)对上述指标有不同程度的改善作用,在体外,11月龄小鼠胸腺淋巴细胞增殖反应明显降低,而其产生的cAMP则明显升高,褪黑素(0.1nmol/L和1μmol/L)对此有反向调节作用,弗司可林(10μmol/L)(腺苷酸环化酶选择性激活剂)能够增强2月龄和11月龄BALB/c小鼠胸腺淋巴细胞内的cAMP水平,褪黑素可部分拮抗此作用,褪黑素的这一作用可被百日咳毒素(1mg/L)完全取消,同时发现褪黑素(1μmol/L和0.1nmol/L)能够明显提高2月龄和11月龄BALB/c小鼠胸腺淋巴细胞的甲硫氨酸脑啡肽含量,这一作用能被硝苯地平(1μmol/L)所阻断,提示褪黑素促进淋巴细胞甲硫氨酸脑啡肽释放可能由Ca~(2+)通道介导。结论:褪黑素对不同月龄小鼠具有免疫调节作用;G蛋白偶联的AC-cAMP信号转导通路和淋巴细胞甲硫氨酸脑啡肽的释放可能是褪黑素发挥免疫调节作用的重要机制之一。     

甘草甜素及地塞米松对体外培养足细胞增殖的影响研究

目的通过建立嘌呤霉素(PAN)诱导小鼠足细胞MPC5损伤模型,观察甘草甜素(GL)及地塞米松(DEX)对足细胞及受损足细胞增殖的影响作用,探讨GL在体外培养足细胞损伤中是否具有类似DEX的防护作用。方法体外培养小鼠足细胞,分为对照组、PAN组、GL组、DEX组、PAN+GL组、PAN+DEX组。对照组采用RPMI-1640培养液培养;PAN组加入终浓度50mg/LPAN;GL组加入不同浓度的GL,终浓度分别为50、100、200、400、600、800mg/L;DEX组加入终浓度1μmol/LDEX;PAN+GL组同时加入PAN(终浓度50mg/L)和GL(终浓度分别为50、100、200、400、600、800mg/L);PAN+DEX组同时加入PAN(终浓度50mg/L)和DEX(终浓度1μmol/L),培养24h及48h,采用MTT检测细胞的增殖活性。结果与对照组相比,PAN组24h及48h时足细胞增殖的光密度(A)值均明显降低(P<0.01),抑制率(IE)分别为55.56%、59.10%;GL组中不同浓度组24h时A值较对照组均明显降低(P<0.01),48h时GL50mg/L组A值与对照组比较差异无统计学意义(P>0.05),其余各组A值较对照组均明显降低(P<0.01);低浓度GL组(100～400mg/L)对足细胞的IE48h较24h明显下降,高浓度GL组(600～800mg/L)对足细胞的IE48h较24h明显升高。DEX组24、48h足细胞增殖A值较对照组均明显降低(P<0.01),48h时足细胞的IE较24h时差异无统计学意义(P>0.05)。PAN+GL组50～600mg/L浓度范围内,24h、48h时足细胞增殖A值均呈剂量依赖性升高(P<0.01),但PAN+GL组800mg/L浓度时足细胞增殖活性不再增加。PAN+DEX组24、48h时足细胞增殖A值均明显升高(P<0.01)。结论 PAN能显著抑制足细胞增殖,GL及DEX对体外培养的正常足细胞增殖有轻微抑制作用,但二者均能促进受损足细胞的增殖,且GL在一定范围内呈剂量依赖性关系,对PAN诱导足细胞损伤具有类似DEX的防护作用。     

乙双吗啉的致突变作用

目的:研究乙双吗啉的遗传毒性.方法:乙双吗啉5,10和15 mg·kg~(-1),腹腔注射观察诱发的小鼠骨髓染色体/染色单体畸变;应用Ames试验观察对测试菌株TA97,TA98,TA100,TA102的诱变作用.结果:乙双吗啉显著诱发小鼠骨髓染色体/染色单体畸变,其诱发的畸变细胞率(ACF)显著增加(P<0.01);在不加S9条件下,乙双吗啉对TA98,TA102有一定的诱发回复突变的作用.结论:乙双吗啉是一种遗传毒物质.     

吗啡对淋巴细胞反应性的影响

本文探讨小鼠或大鼠一次性sc吗啡对分裂原诱导的淋巴细胞增殖反应的影响。结果,吗啡抑制Con A诱导的小鼠或大鼠全血淋巴细胞增殖反应,作用呈浓度依赖关系;但脾淋巴细胞反应性并未明显受损,即使吗啡给药浓度增至50 mg·kg~(-1)。如分离大鼠全血单个核细胞,再观察其对Con A的反应性,则吗啡的抑制活性依然存在,提示全血中的血浆成分不是全血和脾淋巴细胞对吗啡作用敏感性不同的原因。进一步实验发现,纳洛酮10 mg·kg~(-1)预处理可完全拮抗吗啡25 mg·kg~(-1)所致小鼠和大鼠全血淋巴细胞反应性下降,使其恢复至正常水平;而单独给予纳洛酮,对脾和全血淋巴细胞增殖皆无影响。结果表明,吗啡整体给药的免疫调节作用与药物剂量及淋巴细胞的组织来源有关,且其效应通过阿片受体介导。     

Distinction between the in vitro and in vivo inhibitory effects of morphine on lymphocyte proliferation based on agonist sensitivity and naltrexone reversibility.

We have previously reported that administration of a single dose of morphine (25 mg/kg) to rats results in a naltrexone-sensitive suppression of mitogen-stimulated lymphocyte proliferation. To further delineate the site of action of this inhibitory effect, the in vitro and in vivo effects of morphine on mitogen-stimulated lymphocyte proliferation were examined. In vitro, concentrations of morphine exceeding 0.1 mM exhibited a dose-dependent inhibition of Concanavalin A-induced proliferation of both whole blood and splenic lymphocytes. This inhibitory effect of morphine on lymphocyte proliferation was not attenuated by co-incubation with the opioid antagonist naltrexone (0.25 mM). These data indicate that the in vitro inhibitory effects of morphine occur at only high concentrations and are not opioid receptor mediated. In vivo, a dose-dependent inhibition of blood lymphocyte proliferation was also observed 2 h following the subcutaneous injection of morphine. In contrast to these effects, proliferation of splenic lymphocyte cultures was not significantly inhibited by morphine at doses of up to 40 mg/kg. However, following morphine administration, a greater than 90% inhibition of proliferation was obtained in cultures containing either whole blood or Ficoll-separated lymphocytes, indicating that plasma was not a contributory factor in the differential sensitivity of blood and splenic lymphocyte responses to morphine. Moreover, in these experiments, significant inhibition of lymphocyte proliferation occurred at plasma concentrations that were two orders of magnitude less than those required to produce inhibition in vitro. The in vivo inhibition of lymphocyte proliferation by morphine (10 mg/kg) was completely antagonized by pretreatment with naltrexone (5 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alleviation of cyclosporin-induced immune suppression by the cytokine inducer ADA-202-718

ADA-202-718 (ADA) has been shown to augment the production of various cytokines (IL-1, IL-2 and gamma-interferon) by murine T lymphocytes. Administration of 1 mg/kg/day to mice from the time of immunization significantly enhanced delayed-type hypersensitivity responses to sheep red blood cells (SRBC). ADA also inhibited immune suppression in mice given 25 but not 50 mg/kg/day cyclosporin A (CsA). There were, however, few changes compared with vehicle-treated controls in the numbers of splenic regulatory T cell subsets (L3T4+ and Ly2+) following treatment with CsA, ADA or both drugs. On the other hand, splenic lymphocytes from ADA-treated animals exhibited augmented proliferative responses to polyclonal T and B cell mitogens and antigen. ADA (1 mg/kg) also stimulated the splenic IgM plaque-forming cell response to SRBC and prevented CsA (25 mg/kg)-induced suppression of humoral immunity. In vitro, ADA (2 micrograms/ml) inhibited CsA-induced suppression of T cell proliferation, the effect being most marked at lower inhibitory concentrations of CsA. These data illustrate the capacity of ADA to augment or restore T cell responses in experimental animals and are consistent with the view that CsA acts in these experimental in vivo systems by inhibition of cytokine production.     

The influence of prolonged treatment with D-penicillamine on the immune response in Wilson's disease

Summary Humoral and cell-mediated immunity were studied in a group of patients with Wilson's disease not previously treated with D-penicillamine, and in a group of patients treated with the drug for more than two years. The previously untreated patients showed an exaggerated humoral immune response, i. e. increased levels of IgG and, IgM, higher titer of antibodies to Kunin's antigen, and depression of cell-mediated immunity, namely a decreased response to DNCB, decreased lymphocyte transformation after stimulation with Con A, PPD, Candida and streptokinase and a reduced response to streptokinase in the MIF test. After treatment the humoral response returned to normal, and in the case of IgA and antibodies to S. typhi O antigen, it even dropped below normal values. The cell-mediated immune response returned to normal with the exception of lymphocyte transformation by PHA and Candida albicans. In in vitro studies it was found that D-penicillamine had no influence on lymphocyte transformation when PHA and Con A were used as mitogens. With PPD as antigen, lymphocyte stimulation and migration inhibition were inhibited by concentrations of penicillamine ranging from 6 to 1000 µg/ml.     

Immunotoxicity of N,N-diethylaniline in mice: effect on natural killer activity, cytotoxic T lymphocyte activity, lymphocyte proliferation response and cellular components of the spleen

We previously found that N,N-diethylaniline increased the frequency of sister chromatid exchange (SCE) of human lymphocytes to about five times that of the control value, and was as toxic as cyclophosphamide used as a positive control for SCE. To explore whether N,N-diethylaniline affects the function of lymphocytes, we evaluated its immunotoxicity using CBA/N mice. The mice were divided into four groups and received 0, 100, 200, or 400 mg/kg body weight of N,N-diethylaniline by subcutaneous injection. The following items were investigated on days 3 and 7 after injection: body weight, weight of spleen, number of splenocytes, natural killer (NK) and cytotoxic T lymphocyte (CTL) activities, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. The following splenocyte phenotypes were also quantified by flow cytometry: (1) B cells; (2) total T cells; (3) CD4⁺ and CD8⁺ T cells; (4) NK; (5) macrophages and (6) nucleated erythrocytes. The splenic NK and CTL activities in exposed groups significantly decreased compared to the control in a dose-dependent manner and lymphocytes from the 200 and 400 mg/kg groups showed significantly higher spontaneous proliferation. The weight of the spleen and number of splenocytes were significantly higher in exposed groups than in the control. N,N-Diethylaniline also increased the percentages of macrophages, nucleated erythrocytes and B cells in the spleen. On the other hand, N,N-diethylaniline did not affect LPS-stimulated B cell and Con A-stimulated T cell proliferation, or the percentages of NK, total T, and CD4⁺ and CD8⁺ T cells in the spleen or the body weight of mice. The above findings indicated that N,N-diethylaniline selectively inhibited splenic NK and CTL activity and this inhibition was due to decreased NK and CTL functions, but not due to changes in the numbers of splenic NK and T cells.     

Inhibition by fenamates of calcium influx and proliferation of human lymphocytes.

1. Flufenamic and tolfenamic acids have recently been shown to inhibit receptor-mediated calcium influx in human neutrophils. The present work was designed to study the effects of these two nonsteroidal anti-inflammatory drugs on human peripheral blood lymphocyte activation. 2. Peripheral blood mononuclear cells (PBMNCs; containing 90% lymphocytes) were stimulated by mitogen concanavalin A (Con A) or by a combination of an inhibitor of microsomal Ca(2+)-adenosine triphosphatase thapsigargin (TG) and phorbol myristate acetate (PMA). The effects of the two fenamates on cell proliferation were compared with respective changes in calcium metabolism. 3. Flufenamic and tolfenamic acids (10-100 microM) inhibited both Con A and TG + PMA-induced [3H]-thymidine incorporation in a dose-dependent manner. At the same concentration range, the two fenamates inhibited the increase in intracellular free calcium concentration induced by Con A or TG + PMA. This effect was due to inhibition of calcium influx whereas calcium release from intracellular stores remained unaltered. 4. The inhibition of divalent cation influx was confirmed by showing that fenamates inhibited TG + PMA-induced Mn2+ influx. 5. The inhibitory effects of fenamates on PBMNC proliferation and Ca2+ influx were qualitatively similar with those of SK&F 96365, an earlier known inhibitor of receptor-mediated calcium entry. Ketoprofen, a chemically different prostaglandin synthetase inhibitor did not show similar suppressive effects on PBMNCs. 6. The data suggest that flufenamic and tolfenamic acids suppress proliferation of human peripheral blood lymphocytes by a mechanism which involves inhibition of Ca2+ influx and is not related to inhibition of prostanoid synthesis.