氯通道阻断剂对T淋巴细胞增殖的影响

目的探讨氯通道开放在Ｔ淋巴细胞增殖过程中的作用。方法新鲜分离成人外周血Ｔ淋巴细胞,以ＣｏｎＡ诱导其增殖,用［３Ｈ］ ＴｄＲ参入法测定并比较氯通道阻断剂ＤＩＤＳ、ＮＰＰＢ、９ ＡＣ、ＮＦＡ、Ｆｕｒｏｓｅｍｉｄｅ对Ｔ淋巴细胞增殖的作用;测定钙通道阻滞剂ＳＫ＆Ｆ９６３６５、硝苯吡啶以及免疫抑制剂环孢霉素Ａ（ＣｓＡ）对Ｔ淋巴细胞增殖的作用;比较ＤＩＤＳ、ＮＰＰＢ与ＳＫ＆Ｆ９６３６５或ＣｓＡ的相互作用。结果９ ＡＣ,ＮＦＡ,Ｆｕｒｏｓｅｍｉｄｅ对Ｔ细胞增殖无明显抑制作用;ＤＩＤＳ、ＮＰＰＢ呈浓度依赖性地抑制Ｔ细胞增殖,ＩＣ５０分别为１６８２３±３３６和（１２４３５±４２４）μｍｏｌ·Ｌ－１,并显著增强ＳＫ＆Ｆ９６３６５及ＣｓＡ抑制Ｔ细胞增殖的作用,且ＮＰＰＢ增强抑制作用强于ＤＩＤＳ。结论阻断氯通道可抑制ＣｏｎＡ诱导的Ｔ淋巴细胞增殖活化,氯通道开放影响细胞钙运动。     

应用流式细胞仪对实体瘤DNA定量的初步研究

本文用FCDA(Flow Cytometric DNA Analysis)对98例实体瘤患者瘤细胞进行了DNA 含量测定。结果表明,和对照组相比,各种肿瘤细胞DI 值明显增高。其中肺癌、宫颈癌、肝癌、消化道肿瘤、甲状腺癌变化尤为明显(P<0.05);鼻咽癌、腮腺颌下腺瘤和乳腺癌也有增加趋势。肿瘤患者FCM阳性检出率为61.2%。肝癌及癌旁组织细胞,甲状腺癌和其转移灶细胞、腮腺颌下腺肿瘤和其穿制液对比研究,DI 值和倍体分布特征均显示了类似的变化。     

流式细胞仪对人外周血LAK细胞检测的研究

应用流式细胞仪测定14例成人外周血LAK细胞（淋巴因子激活的杀伤细胞）和21例正常成人外周血淋巴细胞的DNA指数（DI），井对细胞增殖周期进行分析。结果显示（1）人外周血LAK细胞的增殖活力明显高于未激活的外周血淋巴细胞；（2）在1～14天培养中，人外周血LAK细胞的增殖活力高，并且稳定；（3）在人外周血LAK细胞中．未找出非整倍体细胞，DI在正常范围。该研究提示：使用人外周血LAK细胞治疗缓解期的急性白血病患者时，LAK细胞不影响流式细胞仪的疗效监测。     

分枝杆菌多糖的制备及对小鼠脾淋巴细胞增殖的影响

目的:制备分枝杆菌多糖并考察其对小鼠淋巴细胞增殖的影响。方法:用苏通培养基培养分枝杆菌,菌体脱脂后,用5种方法提取,比较多糖得率,并用正交试验优化超声提取工艺,再用稀碱提取残渣,粗多糖经Sevag法除蛋白、DEAE-Sepharose Fast Flow层析柱纯化后得到分枝杆菌多糖;用MTT法检测分枝杆菌多糖对小鼠脾淋巴细胞增殖的影响。结果:超声后热水提取多糖得率最高,超声提取的优化工艺是提取时间40 min,固液比为1∶150,超声功率600 W;单因素试验优化的Sevag法除蛋白条件为体积比1∶5,萃取时间为20 min,萃取6次;提取得到的4种多糖组分在6.25~50 mg.L-1范围内均能显著促进脾淋巴细胞的增殖。结论:用本实验工艺成功制备了分枝杆菌多糖,制备物具有刺激小鼠脾淋巴细胞增殖的作用。     

曲马多和吗啡预先给药对小儿静脉血T淋巴细胞增殖及IL-2水平的影响

目的 观察曲马多和吗啡预先给药对小儿静脉血T淋巴细胞增殖及IL-2水平的影响。方法 采集无免疫性疾病的60例门诊小手术患儿外周静脉血5mL,进行全血和外周血单个核细胞（PBMCs）培养。随机分为空白对照组（C组）、吗啡（100ng·mL^-1）组（M组）及曲马多（500ng．mL^-1）组（T组）,每组20例。培养的PBMCs和全血在体外均用相应药物预先作用48h,用刺激剂刺激后测定T淋巴细胞增殖状态及血清白细胞介素2（IL-2）分泌水平。结果 与C组比较,M组cpm值明显降低（P〈0．01）;T组cpm值增高（P〈0．05）。与C组比较,M组IL-2水平明显降低（P〈0．01）;T组IL-2水平增高（P〈0．0S）。结论 吗啡和曲马多对小儿免疫功能有不同的影响。选择曲马多作为小儿术后镇痛的药物更为合适,可以减少对免疫功能的抑制。     

N-乙酰氨基葡萄糖激活Ca2＋信号通路体外诱导小鼠T淋巴细胞增殖

目的N-乙酰氨基葡萄糖（N—acetyl—D—glucosamine,NAc—Glu）是甲壳素的-种重要衍生物。其分子量小,水溶性好,有更独特的生理生化活性,近些年已经引起越来越多的关注。但关于NAc-Glu对T淋巴细胞增殖的影响,国内外罕见报道。本文研究N_乙酰氨基葡萄糖体外作用于小鼠T淋巴细胞诱导淋巴细胞增殖。方法以T淋巴细胞为研究对象,应用MTT法,流式细胞术法考察了NAc—Glu对淋巴细胞的增殖的影响,采用Caz＋探针（Fura-3／AM）、激光共聚焦显微镜和流式细胞仪检测的方法从Ca2＋信号通路入手对NAc—Glu诱导淋巴细胞增殖的机制进行了研究。结果NAc-Glu可以诱导淋巴细胞由G0／G进入S期,使细胞的有丝分裂启动,细胞发生增殖。NAc—Glu作用下的淋巴细胞胞内Ca2＋的浓度显著增加,其中加药6h后淋巴细胞内的Ca2＋浓度最高。24h后有所下降。Ca2＋浓度的改变又影响了CaM和CaN这两种与C矿密切相关的蛋白的表达。     

流式细胞术检测阿司匹林对冠心病患者血小板活化标志物的影响

目的：探讨冠心病患者及其服用阿司匹林前后血小板表面糖蛋白的变化及其意义。方法：采用流式细胞仪（flow cytometry，FCM）测定冠心病患者阿司匹林治疗前后的全血中血小板表面糖蛋白（CD62p／PAC-1）的表达率，采用自身对照分析阿司匹林对血小板表面糖蛋白的影响。结果：冠心病组治疗前的血小板表面糖蛋白PAC-1、CD62p的表达率分别为（10.4±6．2）％和（10．7±7．1）％，较健康对照组明显升高（P＜0．01）．经以阿司匹林为基础的抗血小板治疗后CD62p、PAC-1的表达率下降至（4．3±2．1）％和（4．9±2．4）％（P＜0．01），但仍高于健康对照组。结论：CD62p和PAC-1是血小板活化的敏感和特异指标，阿司匹林能够抑制血小板表面糖蛋白的表达．从而抑制血小板活化，抑制血栓形成。     

胃泌素对淋巴细胞增殖抑制作用的影响

目的 观察胃泌素对淋巴细胞增殖抑制作用的恢复作用.方法 以8种不可预料的中等强度应激作用于大鼠,每天1次共16 d,制备大鼠慢性应激性模型.用MTT法测定淋巴细胞增殖和胃泌素对淋巴细胞增殖的影响.结果 应激时间与MTT比色结果呈直线正相关,外源加入相同浓度的胃泌素可逆转慢性应激对细胞增殖的抑制作用.结论 在一定的时间范围内,慢性应激可抑制淋巴细胞增殖;胃泌索可逆转这种作用.     

枸杞多糖对小鼠T、杀伤T和NK细胞的免疫药理作用及对抗环磷酰胺的免疫抑制作用(英文)

枸杞多糖(LBP)5～10 mg/kg,ip,可以提高小鼠脾脏T淋巴细胞的增殖功能,增强CTL的杀伤功能,特异杀伤率由33％提高到67％.LBP 5mg/kg.ip,可以增强NK细胞的杀伤功能,杀伤率由12.4％提高到18％. LBP 5～10 mg/kg可以对抗环磷酰胺(Cy)对小鼠T、CTL和NK细胞的免疫抑制作用;其中T淋巴细胞的相对增殖指数(RPI)由33％提高到105％,Cy对CTL的抑制率由单用的51％降低到与LBP合用的19％和36％,NK细胞的杀伤率亦由Cy单用的9.5％提到15％和16％.以上结果说明LBP增强了正常小鼠和Cy处理鼠的T细胞介导的免疫反应与NK细胞的活性.     

流式细胞仪测定小鼠网织红细胞比例方法的研究

目的分析和考察流式细胞仪测定小鼠网织红细胞比例实验方法的影响因素,通过明确和优化一些实验参数,制定一个快速、准确和稳定的试验方法。方法参考《欧洲药典》第7版网织红细胞法测定rh EPO体内生物学活性。结果动物个体差异、小鼠在不同剂量下的反应时间及仪器间差异对结果均有一定的影响,但通过优化一些参数和采取一些处理措施可以减小误差。结论优化后的方法能够在较短的时间内得出相对准确和稳定的结果。     

胎盘提取物促淋巴细胞增殖活性及稳定性实验初探

目的从人胎盘组织制备促进免疫细胞增殖提取物并寻求保存提取物的适宜条件。方法通过热处理胎盘匀浆液制备提取物 ,并用溴化四唑蓝染料结合法测定体外促小鼠脾淋巴细胞的增殖活性 ,同时对提取物进行放射性同位素60 Co照射。结果经Lowry′s法测定为每 1g胎盘组织含 2～ 3mg蛋白质 ,其促细胞增殖率 >80 % ,经同位素处理后低温保存数月活性不受影响。结论此法制备的提取物不仅活性高 ,且方法简便、重复性好 ,该提取物作为一种稳定、可靠、明显的促淋巴细胞增殖反应具有上规模开发、生产、应用于临床的价值。     

Assessment of the beryllium lymphocyte proliferation test using statistical process control

Despite more than 20 years of surveillance and epidemiologic studies using the beryllium blood lymphocyte proliferation test (BeBLPT) as a measure of beryllium sensitization (BeS) and as an aid for diagnosing subclinical chronic beryllium disease (CBD), improvements in specific understanding of the inhalation toxicology of CBD have been limited. Although epidemiologic data suggest that BeS and CBD risks vary by process/work activity, it has proven difficult to reach specific conclusions regarding the dose-response relationship between workplace beryllium exposure and BeS or subclinical CBD. One possible reason for this uncertainty could be misclassification of BeS resulting from variation in BeBLPT testing performance. The reliability of the BeBLPT, a biological assay that measures beryllium sensitization, is unknown. To assess the performance of four laboratories that conducted this test, we used data from a medical surveillance program that offered testing for beryllium sensitization with the BeBLPT. The study population was workers exposed to beryllium at various facilities over a 10-year period (1992-2001). Workers with abnormal results were offered diagnostic workups for CBD. Our analyses used a standard statistical technique, statistical process control (SPC), to evaluate test reliability. The study design involved a repeated measures analysis of BeBLPT results generated from the company-wide, longitudinal testing. Analytical methods included use of (1) statistical process control charts that examined temporal patterns of variation for the stimulation index, a measure of cell reactivity to beryllium; (2) correlation analysis that compared prior perceptions of BeBLPT instability to the statistical measures of test variation; and (3) assessment of the variation in the proportion of missing test results and how time periods with more missing data influenced SPC findings. During the period of this study, all laboratories displayed variation in test results that were beyond what would be expected due to chance alone. Patterns of test results suggested that variations were systematic. We conclude that laboratories performing the BeBLPT or other similar biological assays of immunological response could benefit from a statistical approach such as SPC to improve quality management.     

Propionate regulates lymphocyte proliferation and metabolism

• 1.1. The effect of propionate on lymphocyte proliferation and metabolism was investigated. Lymphocytes obtained from human blood and rat mesenteric lymph nodes were utilized.
• 2.2. Propionate at concentrations of 0.04 and 1.0 mmol/1 stimulated the amount of [³H]thymidine incorporated either in cultured human T lymphocytes or rat T and B lymphocytes.
• 3.3. Concentrations of propionate between 2 and 5 mmol/1 caused a marked inhibition of lymphocyte proliferation.
• 4.4. This short-chain fatty acid was metabolized by these cells and produced succinate in significant amounts; however, its oxidation was low.
• 5.5. Propionate did not alter glucose, glutamine and pyruvate utilization and oxidation in incubated rat lymphocytes but increased the formation of lactate and aspartate.
• 6.6. In contrast, propionate inhibited by 50% the synthesis of lymphocyte lipid from [1-¹⁴C]acetate at concentrations of 0.5 and I mmol/1 and reduced by half the incorporation of ³H₂O into lipids at 1 and 5 mmol/1.
• 7.7. The results suggest that inhibition of lipid synthesis is a possible mechanism leading to reduction of lymphocytes proliferation.

     

The effects of saikosaponin on macrophage functions and lymphocyte proliferation.

The effects of saikosaponin-d (ssd), isolated from Bupleurum radix, on phagocytic functions of mouse peritoneal macrophages were investigated after treatment in vitro. The macrophages treated with ssd showed a significant increase in PMA-induced chemiluminescence. An increase in phagocytosis was detected after treatment with saikosaponin-b2 (0.1 microM) for 24 h in vitro, while a suppression of phagocytosis was observed following treatment with saikosaponins (0.5 microM). Treatment with ssd markedly increased the random migration of resident peritoneal macrophages, but did not affect the migration towards FMLP. We further investigated the effect of ssd on proliferative responses of spleen cells and found that ssd, which itself has no mitogenic activity, decreased spleen cell proliferative response to T-cell mitogen, but increased the response to B-cell mitogen.     

The opioid specificity of beta-endorphin enhancement of murine lymphocyte proliferation

Beta-endorphin (beta-end) is a potent analgesic peptide which exhibits a variety of pharmacological activities in the central nervous system (CNS) following binding of its N-terminus to specific opioid receptors. Although C-terminal binding sites for this 31-amino-acid peptide have been characterized in CNS tissue, identification of their possible function has been facilitated by studies of beta-end effects on lymphocyte activities. In this communication, we report a detailed analysis of the opioid specificity of the ability of beta-end to enhance T cell mitogen-induced proliferation in unfractionated murine splenocytes. Intact 31-amino-acid beta-end peptides from several species, including human, camel and rat, enhanced concanavalin A-stimulated [3H]thymidine uptake 50-640% in a dose-dependent, naloxone-irreversible fashion. The presence of the C-terminal amino acids was required for the enhancement activity, since met-enkephalin, alpha- and gamma-endorphin, and human beta-end 1-27 were ineffective. Accordingly, the truncated peptides, human beta-end 6-31 and 18-31, were also able to enhance the Con A response. However, human beta-end 18-31 was consistently not as effective as beta-end 6-31 or the intact 31-residue peptide. These data suggest that although the C-terminus contains the primary active sequence, the N-terminus contributes to the overall potency of the effect. In support of this assertion, N-acetylation, which abolishes opioid binding activity, resulted in a reduced magnitude of enhancement. The data suggest that beta-end interacts with a non-opioid receptor which has specificity characteristics strikingly similar to non-opioid receptors characterized in CNS tissue.     

Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT

Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.     

Pharmacodynamic interaction of recombinant human interleukin-10 and prednisolone using in vitro whole blood lymphocyte proliferation

Prednisolone, a commonly used synthetic corticosteroid, and IL-10, a cytokine under investigation for strong antiinflammatory properties, are being contemplated as a potential joint therapeutic regimen in immune disorders. Their pharmacodynamic interactions were examined in blood from healthy adult male and female volunteers using an in vitro phytohemagglutinin (PHA)-stimulated whole-blood lymphocyte proliferation technique. Isobolograms along with parametric competitive and noncompetitive interaction models were used to determine the nature and intensity of interactions. Single drug effects show prednisolone more efficacious in inhibiting lymphocyte proliferation with an IC(50) of 3.3 ng/mL and I(max) value of 1, signifying complete suppression. Analogous parameters for IL-10 were 16.2 ng/mL for IC(50) and 0.89 for I(max). There were no significant differences in the single drug immunosuppressive effects among genders. Their joint effects showed additive interaction based on isobolographic analysis. Parametric analysis using the competitive interaction model described their interaction as slightly synergistic, while the noncompetitive interaction modeling indicate a small degree of antagonism. Also, the joint effects in females tend to be more antagonistic than males. Concomitant use of prednisolone and IL-10 should thus reflect the net additive responses to concentrations of each agent.     

Suppression of lymphocyte proliferation by hexamethylene diamine

The antiproliferative potential of hexamethylene diamine (HMDA) for mitogen-stimulated splenic lymphocytes was evaluated in vitro at final concentrations of 0.1-16 mM. Addition at the start of culture or after 24 or 48 h of culture decreased the proliferative response to T and B cell mitogens. However, the concentration of HMDA required to cause suppression increased with incubation time. Removal of diamine after 24 h allowed cells to proliferate normally upon reculture with mitogen. Mitogenic responses of cultures containing the potent ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were also inhibited in a time and dose dependent fashion. ODC activity, which was much greater in cultures stimulated with Con A than LPS, was markedly decreased by inclusion of diamine or DFMO in the culture medium. Addition of putrescine to cultures did not reverse the suppressive effects of diamine on proliferation but did restore DFMO-containing cultures to control levels of activity. These results indicate that HMDA does suppress lymphocyte proliferation in vitro by alteration of ODC and polyamine activity. However, comparison of results obtained with DFMO and HMDA suggests that HMDA may act via multiple pathways, only one of which involves inhibition of ODC activity.     

Immunodynamics of methylprednisolone induced T-cell trafficking and deactivation using whole blood lymphocyte proliferation techniques in the rat

Glucocorticoids have diverse effects on various components of the immune system and assessment of such activities in vivo often involves complex techniques and numerous animals. We developed a whole blood technique for determining proliferation rate of lymphocytes in minute amounts of rat blood (5 microL as opposed to a whole rat spleen) (Fasanmade AA, Jusko WJ. J Immunol Methods 1995; 184: 163-167). This method was used in assessment of in vivo T-cell deactivation by methylprednisolone (MP). The blockade of this process by the anti-glucocorticoid, RU 40555, also allows measurement of T-lymphocyte trafficking between vascular and extravascular pools. Blood samples were taken over several hours after iv MP administration to adrenalectomized rats, MP concentrations and lympho-proliferative activities were determined ex vivo after mitogen activation with and without blocking MP with RU 40555. MP disposition was mono-exponential with a t(1/2) of 34 min. The pharmacodynamics (PD) of T-cell trafficking was modeled with a physiological indirect model to generate the IC(50) (0.4 ng/mL) for the inhibitory action of MP on return of T-cells to blood as well as cell trafficking rate constants. The overall suppression of blood T-cells was modeled with an equation which accounts directly for inhibition of the proliferation activity of available blood T-cells with an DC(50) of 0.37 ng/mL. MP produced an initial influx of T-cells to blood within 1 h of infusion, a later marked T-cell depletion with a nadir at 4 h, and return to baseline by 9 h. Lymphocyte deactivation occurred within minutes of MP infusion and returned to baseline in 9 h. MP action was prolonged owing to the low IC(50). This approach for assessing dual features of corticosteroid effects on T-cell trafficking and deactivation allows quantitative PK/PD modeling in small animals such as the rat.