体内外感染PRRSV处理下仔猪脾淋巴细胞增殖活性的变化

为了探讨体内或体外感染猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的仔猪脾淋巴细胞在体外培养的增殖能力,本研究将PRRSV-GXA分离株经Marc-145细胞大量增殖培养,然后体内感染仔猪,接种病毒后分别于第11天、第14天和第21天从活体猪收获脾脏,分离脾淋巴细胞后用ConA和LPS于体外进行诱导增殖.研究结果显示第11天收获的T淋巴细胞增殖能力高于对照组,而B淋巴细胞增殖能力明显下降;第14天收获的T淋巴细胞和B淋巴细胞的增殖能力均极显著低于对照组;第21天收获的B淋巴细胞增殖能力略高于对照组,T淋巴细胞增殖能力略低于对照组.同时,用不同滴度的PRRSV体外感染PRRSV阴性的猪脾淋巴细胞,经ConA和LPS诱导增殖后,发现病毒滴度在103~106 TCID50/mL范围内,能明显提高脾淋巴细胞的体外增殖能力;而病毒滴度在100~102 TCID50/mL范围时,脾淋巴细胞增殖能力低于对照组.本研究结果说明PRRSV体内或体外感染对仔猪脾淋巴细胞增殖活性均有显著的影响,将为临床上PRRS的综合防治提供理论依据.     

甘草甜素对树突状细胞表型及生物学功能的影响

培养小鼠髓系DC2．4细胞,加入LPS（阳性对照组）或甘草甜素,用扫描电镜观察DC的超微结构、流式细胞仪检测DC表面分子MHCII、CD86及CD40的表达、4-氨基安替比林（4-AAP）比色检测DC内酸性磷酸酶活性、ELISA方法检测DC培养上清中IL—12的浓度,体外刺激淋巴细胞增殖实验检测DC对同种异体T淋巴细胞的刺激能力。结果表明,与对照组相比,甘草甜素刺激后,DC表面树突状突起增多,表面分子MHCⅡ、CD86及CD40表达增加,酸性磷酸酶活性下降,培养上清中IL-12浓度升高,刺激同种异体T淋巴细胞的能力也明显增强。结果表明,甘草甜素能够促进小鼠髓系DC2.4表型及功能的成熟。     

大豆异黄酮对电离辐射小鼠淋巴细胞的防护作用

目的研究大豆异黄酮(SI)对小鼠淋巴细胞的辐射防护作用。方法24只雄性昆明小鼠,随机分为正常对照组、辐射对照组和辐射补充0.5%SI组,喂养2周后,4.0Gy照射。照射后24 h处死小鼠,取血、胸腺和脾脏分离淋巴细胞,进行血淋巴细胞计数、观察DNA损伤情况;培养胸腺和脾脏淋巴细胞,检测3H-dT掺入量,观察淋巴细胞的增殖能力,计算淋巴细胞的增殖指数。结果辐射使小鼠胸腺和脾脏淋巴细胞数明显减少、胸腺淋巴细胞增殖能力和脾脏淋巴细胞转化指数降低、血淋巴细胞DNA损伤增加,这些变化均具有统计学意义;补充SI可降低胸腺和脾脏淋巴细胞数的减少幅度,降低胸腺淋巴细胞增殖能力和脾脏淋巴细胞转化指数下降幅度,减少辐射对血淋巴细胞DNA损伤程度,其中SI对胸腺淋巴细胞增殖能力和对血淋巴细胞DNA损伤程度的防护作用与辐射对照组相比有统计学意义。结论大豆异黄酮可对小鼠的血、胸腺和脾脏淋巴细胞有一定的辐射防护作用。     

慢性肾衰病人血清和红细胞抗氧化能力的ESR研究

用电子自旋共振(ESR)自旋捕集技术研究了正常人和肾衰病人血清和红细胞对黄嘌呤 黄嘌呤氧化酶体系产生的氧自由基的作用.结果发现:(1)正常人血清和红细胞能够有效地清除超氧阴离子自由基(O_2~-),而肾衰病人血清和红细胞清除O_2~-的能力明显比正常人血清和红细胞低;(2)正常人血清能有效地把黄嘌呤 黄嘌呤氧化酶体系产生的O_2~-转化为·OH,病人血清在这方面与正常人血清有显著性差异.     

甲2巨球蛋白制剂对正常人外周血淋巴细胞周期的影响

采用外周血淋巴细胞姐妹染色单体差别法(SCE)检测技术,研究甲_2巨球蛋白制剂α_2M对人外周血淋巴细胞周期的影响,在人外周血淋巴细胞培养中加入α_2M制剂浓度在≤0.5mg/ml培养液时,第一次细胞周期(M_1)、第二次细胞周期(M_2)、第三次细胞周期(M_3)、细胞增殖速度指数(PRI)改变不大。在加入的α_2M制剂浓度(?)1.0 mg/ml培养液时,M_1百分数增加(r=0.977,P<0.001)、M_2百分数也随α_2M浓度递增而增高(r=0.956,P<0.001)而M_3百分数则随α_2M浓度递增而减少(r=-0.979,P<0.001)、细胞增殖速度指数则随α_2M浓度递增而减少(r=-0.983,P<0.01),表明在人外周血淋巴细胞离体培养时,当每毫升培养液中α_2M浓度等于或大于1mg时,细胞分裂周期延缓。讨论了它的意义和可能作用的环节。     

连翘酯苷对小鼠脾脏淋巴细胞体外增殖与分泌功能的影响

目的分析连翘酯苷（FS）对小鼠脾脏T和B淋巴细胞增殖、分泌NO和TNF-α的影响,初步探讨其免疫调节作用机制。方法无菌操作分离小鼠脾脏,制备脾脏细胞并用含10%胎牛血清的RPMI 1640培养,在培养液中分别加入刺激剂刀豆蛋白（ConA）和脂多糖（LPS）以及不同浓度40、80、160μg/mL的FS共培养不同时间,采用MTT法检测T和B淋巴细胞的吸光度变化,ELISA和Griess法分别检测细胞分泌TNF-α和NO的水平。结果低浓度和中浓度FS对ConA诱导T淋巴细胞24 h和48 h后细胞增殖和存活率明显提高,诱导时间延长至72 h后FS明显抑制细胞转化;低浓度FS对LPS诱导脾脏B淋巴细胞24 h后细胞增殖和生存率显著提高;FS促进小鼠脾脏T和B淋巴细胞分泌NO;FS促进B淋巴细胞分泌TNF-α,中浓度FS促进T淋巴细胞分泌TNF-α而高浓度反而抑制其分泌。此外,FS对环磷酰胺（CY）处理小鼠的脾脏淋巴细胞体外增殖有明显影响,对细胞NO分泌影响不显著。结论结果提示FS可能通过影响小淋巴细胞增殖和细胞因子分泌而调节免疫细胞功能。     

青藤碱对人外周血CD4^＋T淋巴细胞增殖和细胞内Ca^2＋浓度影响的体外研究

目的探讨青藤碱（SIN）对人外周血CD4^＋T淋巴细胞增殖和细胞内Ca^2＋浓度的体外影响及其效应机制。方法建立体外人外周血CD4^＋T淋巴细胞模型,分别作以下处理：（1）空白对照组;（2）环孢素（CsA）组（50ng／m1）;（3）低浓度SIN组（10μmol／1）;（4）中浓度SIN组（200μmol／1）;（5）高浓度SIN组（1000μmol／1）。分别用MTT比色法和流式细胞术（FCM）检测CD4^＋细胞增殖和细胞内Ca^2＋荧光强度。采用方差分析比较各组间差异的统计学意义。结果（1）高浓度SIN组、中浓度SIN组与其他各组细胞增殖抑制率存在差异（F=1444．228,P=0．000）;（2）FCM检测细胞内Ca^2＋浓度结果：中浓度SIN组、高浓度SIN组与其他各组差异有统计学意义（F=479．055,P=0．000）;（3）经SIN处理后,人外周血CD4^＋T淋巴细胞增殖抑制率和细胞内Ca^2＋浓度之间存在负相关r=-0．836,P=0．005）。结论SIN能浓度依赖性地抑制人外周血CD4^＋T淋巴细胞增殖和细胞内Ca^2＋浓度升高,人外周血CD4^＋T淋巴细胞增殖抑制率和细胞内Ca^2＋浓度之间存在显著性负相关。     

雪灵芝粗多糖对小鼠免疫细胞增殖与功能的体外激活作用

为观察雪灵芝粗多糖(Arenaria kansuensis crude polysaccharide,AKCP)对体外培养的小鼠脾淋巴细胞、NK细胞和腹腔巨噬细胞增殖与功能的影响。以不同浓度AKCP作用于体外培养的上述细胞48 h,采用中性红吞噬实验及NO释放实验检测巨噬细胞功能,MTT法检测脾淋巴细胞增殖及NK细胞杀伤活性,流式细胞术检测脾淋巴细胞CD3~+、CD4~+、CD8~+亚群,ELISA法检测脾细胞培养上清中IL-2和IFN-γ水平。结果显示,AKCP各浓度组小鼠腹腔巨噬细胞的吞噬活性和NO释放量、脾淋巴细胞刺激指数及培养上清中IFN-γ水平、NK细胞杀伤活性均高于空白对照组(P0.05);AKCP中浓度组脾淋巴细胞CD3~+、CD4~+亚群及培养上清中IL-2水平也明显升高(P0.05)。提示AKCP对小鼠免疫细胞的增殖与功能具有体外激活作用。     

豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响

利用3H-TdR放射性来测定T淋巴细胞增殖强度的方法,研究了豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响.结果表明,豆腐柴根提取物在一定浓度范围内可促进刀豆蛋白(ConA)诱导T淋巴细胞发生增殖反应,其中2μg.mL-1提取物浓度效果最为明显.该研究为合理开发利用豆腐柴这一野生药物资源提供了科学的实验依据.     

用碱性连二亚硫酸钠水溶液产生超氧阴离子自由基

产生超氧阴离子自由基(O_2~-)虽有多种方法,例如黄嘌呤-黄嘌呤氧化酶法、碱性二甲基亚砜法等,但简单易行的方法迄今未见有介绍。本文报道用氧饱和的碱性连二亚硫酸钠(Na_2S_2O_4)水溶液产生O_2~-,方法简易,O_2的浓度稳定。     

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.     

Mechanism of suppression of human peripheral blood lymphocyte proliferation by platelet activation factor

The influence of the phospholipid platelet activation factor (PAF) was studied on PHA-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that influencing on the whole population of lymphocytes PAF suppressed proliferation mainly of T-cells. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the suppression of lymphocyte proliferation induced by PHA. Pretreatment of lymphocytes with PAF antagonist--BL 8705 almost completely blocked the suppression of lymphocyte proliferation induced by PHA.     

Synthesis, immunological activities, and scavenging ability toward superoxide anion of (1-->3)-beta-D-pentaglucoside and its epoxyalkyl derivatives

The epoxyalkyl (1-->3)-beta-D-pentaglucosides 2 and 3 were synthesized in order by acetylation, glycosidation, oxidation, and deacetylation of 1. The immunological activities (superoxide anion production activity, phagocytic activity, and lymphocyte proliferation) and scavenging ability toward superoxide anion of (1-->3)-beta-D-pentaglucoside (1) and its epoxyalkyl derivatives (2 and 3) were investigated. Superoxide anion released from human blood monocytes was measured by the reduction of ferricytochrome c. Phagocytosis by peritoneal macrophages was detected through a teal ingesting that measured the chicken red blood cells (CRBC). Lymphocyte proliferation was determined by the MTT method. The scavenging ability of 1, 2, and 3 toward superoxide anions was evaluated by means of chemiluminescence (CL). The results showed that 2 and 3 had a little higher immunological activity and scavenging ability toward superoxide anion than 1, which indicated that the reducing end of the oligoglucosides was quite important for maximum biological activity.     

Induction of proliferation of human circulating monocytes in vitro by lectin-induced factor(s) from lymphocytes

Human peripheral blood monocytes, which have been considered to be non-dividing cells, were induced to proliferate in vitro by soluble mediator(s) from lectin-activated human lymphocytes. The lectin-induced factor from lymphocytes increased both the number of nuclei of cultured monocytes and [3H]-thymidine incorporation into the monocytes. The molecular weights of the soluble factor(s) that promote growth of monocytes were in the range of 20,000-70,000 daltons with two peaks.     

Phosphorylation of Histone H2AX on Ser 139 and Activation of ATM During Oxidative Burst in Phorbol Ester-Treated Human Leukocytes

Oxidative burst is a defense mechanism used by specialized phagocytes such as granulocytes or monocytes to kill the invading microorganisms through generation of superoxide anions. Oxidative burst also results in DNA damage of the phagocytes. Phagocytes are terminally differentiated and some of very short life-span cells. We could find no reports whether oxidative burst-mediated DNA damage triggers in such cells histone H2AX-Ser139 phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM), the signals otherwise used to activate DNA repair and checkpoint pathways in proliferating cells. We now present the evidence that induction of oxidative stress in human peripheral blood leukocytes by phorbol myristate acetate (PMA) was associated with intense phosphorylation of histone H2AX and with ATM activation, seen already 60 min after exposure to PMA. The modifications of H2AX and ATM in individual granulocytes, monocytes and lymphocytes were detected prior to caspases activation and thus were unrelated to induction of apoptosis. A large intercellular variation in response was observed, and only a fraction of cells in these subpopulations showed H2AX and ATM modifications. The data are compatible with the earlier observations of DNA damage during oxidative burst and suggest that even in terminally differentiated cells that have a short life-span, DNA damage triggers recruitment of the DNA repair machinery. The observed H2AX phosphorylation in lymphocytes may reflect their DNA damage by the superoxide ions propagating from the neighboring granulocytes and/or monocytes.     

Effects of phospholipid platelet activation factor on proliferation of B-lymphocytes of the peripheral blood of healthy donors and patients with bronchial asthma

The influence of the phospholipid platelet activation factor (PAF) was studied on pokeweed mitogen (PWM)-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that PAF exerts dose-dependent activation with following suppression mainly on B-cells after its action on the whole population of lymphocytes activated by PWM. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the lymphocyte activation induced by PWM. Indomethacin inhibits lymphocyte proliferation activation induced by PAF. The results indicate that PAF has an effect on the IgE and IgG synthesis by blood B-lymphocytes in patients with bronchial asthma.     

柱状噬纤维菌免疫后兴国红鲤免疫细胞数量变化的研究

用柱状噬纤维菌灭活菌苗免疫兴国红鲤,于免疫后2周,4周分析对其外周血液和胸腺,脾脏,头肾和体肾中的免疫细胞数量变化进行了测定,结果显示：外周血液中,淋巴细胞有显著的增加,粒细胞和单核细胞有明显的下降,胸腺中淋巴细胞数目略有增加,脾脏中淋巴细胞有显著的增加,而粒细胞和单核细胞正相反,头肾中淋巴细胞有较多的增加,粒细胞数目增加不明显,单核细胞有大幅下降,体肾中,粒细胞和淋巴细胞大幅上升,单核细胞下降。     

Tumor necrosis factor-α can induce Langhans-type multinucleated giant cell formation derived from myeloid dendritic cells

The formation of the rich cellular features of MGCs, where the nuclei are arranged circularly at the periphery of the cell (morphologically epithelioid; Langhans-type), is assumed to be associated with any granulomatous disease. The mechanism by which TNF controls the formation of human MGCs in vitro was investigated, focusing on the effect of the TNF-neutralizing antibody. Peripheral blood monocytes were isolated with mAb-coated immunologic magnetic beads and cultured for 10 days in the presence of 20 ng/mL GM-CSF and 10 ng/mL IL-4. These cells were further incubated in the presence of TNF-α with/without its blockade antibodies for 14 days. Myeloid DCs can be generated from peripheral blood monocytes, and both IL-4 and GM-CSF can provide sufficient stimulus for their differentiation. The formation of MGC can be induced in the presence of TNF-α. This reaction was prohibited by the presence of the TNF-neutralizing antibody but not by the presence of anti-TNF receptor II antibody. The activation of Rho and focal adhesion kinases induced by TNF-α stimulation might be linked to cell assembling and the formation of Langhans-type MGCs. MGCs can produce only small amounts of superoxide anions compared to isolated macrophages such as myeloid DCs.     

Effects of blood monocytes and lymphocytes on progesterone secretion by granulosa cells in the pig

We studied the effects of autologous and nonautologous co-cultures of porcine blood monocytes and lymphocytes with granulosa cells on progesterone secretion. Eight prepubertal crossbred gilts were ovariectomized, and the granulosa cells were collected, plated at 2.5 x 10(5) cells/ml and allowed to attach. Blood was obtained from the same eight gilts, and the mononuclear cells were separated by density gradient centrifugation. Monocytes were separated from lymphocytes by adherence to plastic. Adherent monocytes, lymphocytes and a 1:1 mixture of monocytes + lymphocytes were added to granulosa cell cultures and incubated for 48 h. Progesterone secretion into the media was measured. In addition, blood cell alloreactivity was studied in these co-cultures by measuring uptake of (3)H-thymidine. The co-culture of adherent monocytes or monocytes + lymphocytes with granulosa cells increased (P <.05) progesterone secretion as compared with granulosa cells cultured alone. However, co-culture of lymphocytes with granulosa cells did not have a significant effect. No difference was observed between autologous and nonautologous cell cultures in blood cell proliferation or granulosa cell progesterone secretion. In conclusion, blood monocytes influence progesterone secretion by granulosa cells. In addition, there was no difference in the ability of autologous and nonautologous blood cells to stimulate progesterone secretion by granulosa cells. No alloreactivity was observed using nonautologous immune cells with granulosa cells.     

Differential effects of human blood monocytes on the growth of human tumour cell lines in vitro

Summary Monocytes and macrophages have been shown to be cytotoxic towards tumour cells in vitro. However, although tumour-associated monocytes and macrophages are now widely accepted to contribute a relatively high proportion of the cellular infiltrate of experimental and human solid carcinomas, a cytotoxic/cytostatic effector function for these cells in vitro or in vivo has yet to be conclusively demonstrated. In the present study, we show that non-activated peripheral blood monocytes co-cultured with tumour cells across a semi-permeable membrane release soluble factors that modulate the growth of tumour cells in contrasting ways. After Nycoprep 1.068 separation, non-activated peripheral blood monocytes enhanced the in vitro proliferation of HT29 colon adenocarcinoma cells but inhibited T47D breast carcinoma cell replication; peripheral blood lymphocytes were incapable of mediating these effects. In contrast, peripheral blood monocytes activated by interferon caused a pronounced inhibition of both HT29 and T47D cell proliferation.