聚酯型儿茶素对小鼠淋巴细胞增殖和凋亡的影响

目的 :研究聚酯型儿茶素 (theasinesin ,TS)对小鼠淋巴细胞增殖和凋亡的影响。方法 :采用MTT法研究TS对小鼠脾细胞增殖的影响 ,采用形态学检测、DNA琼脂糖凝胶电泳及FACS分析方法研究TS对小鼠胸腺细胞自发凋亡及地塞米松诱导脾细胞凋亡的影响。结果 :5 0、15 0、5 0 0mg·L- 1TS对小鼠脾淋巴细胞增殖有明显促进作用。小鼠胸腺细胞体外培养 2 0后 ,琼脂糖凝胶电泳出现典型的DNAlad der,FACS图上出现典型的亚二倍体凋亡峰。 5 0、15 0、5 0 0mg·L- 1TS作用后 ,随药物剂量增加DNAladder减弱 ,凋亡峰减小 ,细胞凋亡率从 19.87%分别减为 12 .14%、9.4 9%、6 .71%。但不同浓度TS对地塞米松诱导的小鼠脾细胞凋亡无明显影响。结论 :聚酯型儿茶素可明显促进小鼠脾淋巴细胞增殖 ,同时可抑制小鼠胸腺细胞自发凋亡 ,两者总的效应是一致的。     

Comparison of lectin fromPseudixus japonicus and concanavalin a on lymphocytes proliferation and cytotoxicity

Pseudixus japonicus agglutinin (PJA) was isolated. And its characteristics were compared with those of concanavalin A (Con A). PJA is a glycoprotein composed of 49.3% carbohydrate and 50.7% protein which had relatively high percentages of glutamic acid, aspartic acid and phenylalanine residues. The hemagglutinating activity of PJA was approximately one-eighth of that of Con A when tested with mouse erythrocytes. PJA failed to stimulate the proliferation or transformation of human and mouse lymphocytes in contrast to Con A. PJA and Con A showed cytotoxicities against SNU-1 (human stomach cancer cells), SNU-C1 (human colon cancer cells) and mouse Sarcoma 180 cells when tested by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. The antitumor activity of the lectinin vivo was also tested in Sarcoma 180 bearing mice. There was no significant difference in prolongation of life span of the mice after the treatment with PJA and Con A for 10 consecutive days.     

咖啡酸苯乙酯对小鼠CD3~+T细胞体外活化和增殖的影响

目的研究咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)对小鼠CD3+T淋巴细胞活化及增殖的影响,探讨其作用机制。方法无菌分离小鼠淋巴结淋巴细胞,制备淋巴细胞悬液。在淋巴细胞活化实验中,分别与不同浓度的咖啡酸苯乙酯预先孵育2h后,加入促分裂原伴刀豆蛋白A(ConA),运用荧光抗体染色技术结合流式细胞术,8h后,检测T淋巴细胞早期活化标记分子CD69的表达情况;24h后,检测T淋巴细胞中期活化标记分子CD25的表达情况;在淋巴细胞增殖试验中,运用CFDA-SE标记法检测T淋巴细胞刺激48h后的增殖情况。结果CAPE(0·5、1、5、10mg·L-1)能够明显地抑制ConA刺激的小鼠淋巴细胞活化,且呈浓度依赖性;CAPE(0·5、1、5、10mg·L-1)能够明显地抑制ConA刺激的小鼠淋巴细胞增殖,且呈浓度依赖性。结论CAPE对小鼠CD3+T淋巴细胞的体外活化和增殖具有抑制作用,其作用机制可能通过同时抑制PLC-γ信号途径和MAP激酶途径,由此推理CAPE是一种潜在的有效的免疫抑制剂。     

Effects of dialkoxylphenyl compounds with oxime group on macrophage function and the proliferation of lymphocytes

Dialkoxyphenyl compounds have been reported to possess anti-inflammatory activity through inhibition of phosphodieseterase (PDE) type IV. In this study, a series of derivatives of dialkoxyphenyl compounds with an oxime group, which is generally known to be one of the biologically active functional groups, were prepared and evaluated for their ability to inhibit the production of inflammatory mediators in activated macrophages and the proliferation of lymphocytes. The structure-activity relationship (SAR) study with 12 compounds on tumour necrosis factor (TNF)-alpha inhibition, analysed by the oxime geometry and different size of spacers between the oxime and phenyl group, indicated that there might be at least three possible hydrogen bonding sites in the inhibitor binding pocket of PDE IV. Of them, compound 6 clearly displayed the highest inhibitory effect on in-vitro TNF-alpha production from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 6 also suppressed in-vivo TNF-alpha release from LPS-primed mice, a level comparable with that of the standard PDE IV inhibitor, rolipram. In addition, oxime compounds also significantly inhibited both nitric oxide production from activated RAW264.7 cells and T lymphocyte proliferation elicited by concanavalin A but not IL-2. The data suggest that the oxime group may act as a functional group, capable of interacting with the inhibitor-binding pocket of target PDE IV. Therefore, it is conceivable that compound 6 may have the potential either to be developed as a new anti-inflammatory drug or to be used to develop more potent analogues.     

Quantification of uptake of liposomal carboxyfluorescein by professional phagocytes in-vitro. A flow microfluorimetric study on the J774 murine macrophage cell line

Unilamellar egg phosphatidylcholine/cholesterol liposomes containing carboxyfluorescein were prepared by an ether injection method. The ability of cells of the J774.2 murine macrophage cell line to incorporate the liposomal fluorophore during incubation at 37 degrees C was measured by flow microfluorimetry. Liposomes incorporating additional phosphatidylserine or phosphatidic acid were taken up much more avidly than those lacking these phospholipids and the greatest uptake of carboxyfluorescein was observed with the phosphatidylserine species. Calculation of the number of liposomes taken up, greater than or equal to 0.2% of the number given, showed that this was an inefficient process. However these uptake data support previous findings based on the intracellular bactericidal activity of liposomal antibiotics determined in an identical in-vitro system.     

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.     

吗啡及纳洛酮对淋巴细胞体外增殖的作用(英文)

目的:研究吗啡对不同淋巴细胞增殖的作用及纳洛酮的影响.方法:观察吗啡对未成熟的、静止的及活化的脾脏淋巴细胞体外增殖影响及纳洛酮的阻断作用.结果:吗啡(1×10~(-10)—1×10~(-6)mol L~(-1))能增加Con A诱导的T-细胞的增殖,1 μmol L~(-1)还能促进LPS诱导的B-细胞的增殖,同时这些增强作用都能被纳洛酮50μmol L~(-1)阻断,纳洛酮单独亦能促进活化T-细胞的增殖.而吗啡1×10~(-10)—1×10~(-5)mol L~(-1)对静止的脾脏淋巴细胞及Con A活化的胸腺淋巴细胞的增殖都无影响.但是吗啡1mmol L~(-1)能广泛抑制静止的、LPS活化的脾脏细胞及Con A活化的胸腺,脾脏淋巴细胞增殖,且都不能被纳洛酮阻断.结论:吗啡对活化T-和B-细胞的促进作用是由细胞表面的阿片受体介导的,此阿片受体随着淋巴细胞的成熟和活化而变化,而吗啡1 mmol L~(-1)对淋巴细胞增殖的抑制作用却不是由经典的阿片受体介导的.     

Effects of tartrazine on proliferation and genetic damage in human lymphocytes

The color additive, tartrazine (TRZ), is widely used in food products, drugs and cosmetics. Genotoxicity of TRZ and its metabolites has not been investigated in detail in the presence and absence of a metabolic activator (S9 mix) in human. Therefore, the aim of this study is to investigate the cytotoxic and genotoxic effects of TRZ and its metabolites on cultured human lymphocytes by using chromosome aberration (CA) and micronucleus (MN) tests. Cultures were treated with 625, 1250 and 2500?μg/ml of TRZ in the presence and absence of S9 mix. TRZ showed cytotoxic activity at the highest concentration due to significant decrease in mitotic index (MI) in the absence of S9 mix when compared with solvent control. TRZ and metabolites significantly increased the CAs and aberrant cells in the presence and absence of S9 mix at the higher concentrations. Increased MN values in cultures with and without S9 mix were found to significantly at the highest concentration when tested. Our results indicated that while both TRZ and its metabolites have genotoxic potential on human lymphocyte cultures with and without S9 mix, TRZ can induce cytotoxicity at the highest concentration in culture without S9 mix under the experimental conditions.     

紫杉醇对小鼠T细胞CD69、CD25表达及增殖作用的流式细胞术分析

目的研究紫杉醇对多克隆刺激剂作用下小鼠T细胞CD69、CD25表达及增殖的影响,并探讨其机制。方法利用荧光标记的单克隆抗体染色结合流式细胞术,检测多克隆刺激剂佛波醇酯(PDB)或刀豆蛋白(ConA)刺激下小鼠T细胞表达活化抗原CD69、CD25的百分率;以羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDASE)标记技术结合流式细胞术分析PDB+Ion(Ionomycin)或ConA刺激下T细胞增殖指数。结果PTX无论对ConA或PDB刺激组T细胞CD69表达均无抑制作用(P>0.05),而对T细胞CD25表达有明显抑制作用(P<0.05),且呈剂量依赖关系;PTX无论对ConA或PDB+Ion刺激组T细胞增殖均有抑制作用(P<0.05),且呈剂量依赖关系。但在ConA及PDB+Ion刺激初立即或是24h后加入不同浓度的PTX,PTX抑制T细胞增殖作用无差异(P>0.05)。结论PTX可明显抑制多克隆刺激剂诱导的T细胞中后期活化及增殖,其作用机制可能与早期活化相关蛋白PTK及PKCθ无关,而与PKCθ下游活化信号相关。     

Effects of macrocyclic trichothecene congeners on the viability and mitogenesis of murine splenic lymphocytes

The effects of several congeners of the macrocyclic class of trichothecene mycotoxins on murine splenic cells in vitro were investigated. The mycotoxins were roritoxin B, myrotoxin B, roridins A, D and E, baccharinoids B4, B5 and B12, 16-hydroxyverrucarin A, and verrucarins A and J. Lymphocytes from CD-1 mice were cultured with each of the mycotoxins for 48 h to assess cytotoxicity. The maximum effect of various trichothecenes produced on cells occurred at concentrations ranging from 10(-6) to 10(-4) M. Mycotoxins had no effect at concentrations ranging from 10(-12) to 10(-7) M. The mitogenic stimulants concanavalin A, lipopolysaccharide, phytohemagglutinin, and pokeweed mitogen were added to splenic lymphocyte cultures along with varying concentrations of selected mycotoxins. Blastogenesis was inhibited at concentrations 2-5 orders of magnitude lower than those which produced lethality on resting lymphocytes.     

Effects of sinefungin and 5'-deoxy-5'-S-isobutyl-adenosine on lipopolysaccharide-induced proliferation and protein N-methylation of arginyl residues in murine splenic lymphocytes.

The effects of sinefungin (SF) and 5'-deoxy-5'-S-isobutyl-adenosine (SIBA) on lymphoproliferation and protein N-methylation of arginyl residues were investigated in splenic lymphocyte culture in vitro. Both SF and SIBA produced a dose-related inhibition of the lymphoproliferative response to lipopolysaccharide (LPS). The time-course of the inhibition indicated that SIBA had a more inhibitory effect than that of SF at short incubation periods, but SF showed more inhibition when the incubation was prolonged. The effects of SF and SIBA on the lymphoproliferative response corresponded with their effects on the protein N-methylation of arginyl residues by protein methylase I.     

可溶性CD40基因修饰树突状细胞对实验动物淋巴细胞增殖及胞毒活性的影响

目的探讨可溶性CD40-增强绿色荧光蛋白C(sCD40-EGFP)修饰树突状细胞(DC)对实验动物免疫功能的影响,为利用DC诱导供体特异性移植免疫耐受提供实验依据。方法应用基因工程技术构建CD40胞外区与EGFP重组载体质粒pEGFP-N1/sCD40,采用脂质体转染法,将其导入DC2.4细胞株。采用荧光分光光度计和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定转染质粒pEGFP-N1/sCD40的DC培养上清;将经未修饰DC细胞及sCD40-EGFP及空载体修饰DC经腹腔注射致敏Balb/c小鼠,取单个核细胞作为反应细胞,分别以不同组DC为刺激细胞,行混合淋巴细胞培养,采用MTS比色法检测细胞增殖,乳酸脱氢酶法测定细胞毒活性。结果sCD40分子与EGFP融合基因载体构建成功,在树突状细胞中获得表达,并分泌至上清;sCD40-EGFP融合蛋白对不同组DC致敏或未致敏小鼠的同种细胞刺激的增殖反应均有明显的抑制作用,sCD40-EGFP基因修饰DC诱导不同DC致敏组小鼠淋巴细胞增殖反应均明显降低,sCD40-EGFP融合蛋白对各实验组淋巴细胞胞毒活性有显著的抑制作用,sCD40-EGFP基因修饰DC致敏组小鼠淋巴细胞胞毒活性较其余实验组明显降低。结论稳定表达sCD40-EGFP融合蛋自的DC致敏可显著降低同种小鼠淋巴细胞的增殖反应,并能诱导淋巴细胞对靶细胞的胞毒活性降低。     

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.     

低氧暴露对小鼠脾淋巴细胞增殖和凋亡的影响

目的 研究低氧暴露诱导淋巴细胞数量减少与细胞增殖和凋亡的关系。方法 从C57BL/6小鼠脾中分离淋巴细胞,将淋巴细胞分别在低氧（1%O₂）和常氧（21%O₂）环境中培养12、24、48 h。用流式细胞术检测T、B淋巴细胞数量;羧基荧光素乙酰乙酸和AnnexinⅤ-FITC/PI方法分别检测淋巴细胞的增殖和凋亡;扫描电镜观察淋巴细胞形态;实时荧光定量逆转录PCR和Western blot检测凋亡相关因子鼠源B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2同源拮抗剂-杀伤蛋白（Bak）和胱天蛋白酶-3（caspase-3）的mRNA和蛋白表达水平。结果 低氧暴露12、24、48h,可降低T、B淋巴细胞数量,抑制淋巴细胞增殖,促进细胞凋亡;扫描电镜观察显示,同一时间下,低氧组淋巴细胞更早出现凋亡特征形态学变化;低氧暴露12、24、48 h,淋巴细胞Bak和caspase-3的mRNA和蛋白表达水平均上调,而bcl-2的mRNA和蛋白表达水平均下调。结论 低氧暴露通过抑制淋巴细胞增殖、促进凋亡,从而介导淋巴细胞数量下降。     

The use of flow cytometric methods in acute and long-term in vitro testing.

One principal demand for in vitro screening for toxic effects is the ease of performance and the high throughput of test methods. Flow cytometry offers the possibility to study several parameters simultaneously, e.g. cell cycle modulation, mode of cell death, activity of mitochondria. Aim of the present study was to assess the suitability of flow cytometry for the determination of cytotoxicity of test chemicals. Six chemicals chosen from the MEIC list (acetaminophen, isoniazid, paraquat, malathion, digoxin and 2,4-dichlorophenoxy acetic acid) were tested in HepG2, AAH-1, YAC-1 cells and human lymphocytes. Chemicals were applied for 24, 48 h or 28 days. The phases of the cell cycle were determined and the induction of apoptosis and necrosis was demonstrated by annexin binding, analysis of mitochondrial membrane potential and DNA strand breaks. The results of the present study show that flow cytometric methods are well suited to screen for the cytotoxicity of chemicals, both in adherent cells and cells grown in suspension.     

Effects of spider Macrothele raven venom on cell proliferation and cytotoxicity in HeLa cells

AIM: To examine the effect of venom from the spider Macrothele raven on cell proliferation and cytotoxicity in human cervical carcinoma, HeLa cells. METHODS: Morphological and biochemical signs of apoptosis appeared using acridine orange-ethidium bromide (AO/EB) staining. Marked morphological changes in HeLa cells after treatment with spider venom were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell proliferation and cytotoxicity were determined by [methyl-3H] thymidine assay ([3H]TdR) and lactate dehydrogenase (LDH) release, respectively. DNA fragmentation and cell cycle distribution were monitored using flow cytometry. In addition, Western blot analysis was used to evaluate the level of caspase-3 expression. In vivo examination of the inhibition of the size of tumors in nude mice treated with spider venom was measured. RESULTS: Marked morphological changes were observed using AO/EB staining, SEM and TEM assay. Spider venom at concentrations of 10-40 mg/L caused dose- and time-dependent inhibition of HeLa cell proliferation. The ratio of apoptosis and necrosis increased. The activity of caspase-3 was upregulated after spider venom treatment. In vivo study of tumor size revealed that tumors significantly decreased in size from controls to tumors treated for 3 weeks with spider venom (P<0.05). CONCLUSION: The inhibition of HeLa cells by the venom of the spider Macrothele raveni was carried out in three ways: induction of apoptosis, necrosis of toxicity damage and direct lysis. Spider venom is a novel anti-tumor material both in vitro and in vivo.     

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.     

Tetranactin, a macrotetrolide antibiotic, suppresses in vitro proliferation of human lymphocytes and generation of cytotoxicity

Tetranactin, a hydrophobic cyclic antibiotic produced by Streptomyces aureus, has previously been shown to suppress in vitro activation of rat lymphocytes by concanavalin A as well as the onset of experimental autoimmune uveoretinitis in Lewis rats. Here we report the effects of tetranactin on human T and NK lymphocytes in vitro. Tetranactin, at concentrations up to 100 ng/ml, was not toxic to human lymphocytes but completely abrogated the proliferation of human T lymphocytes in response to allogeneic cells in mixed lymphocyte cultures. Tetranactin also blocked the initiation of proliferation in response to interleukin-2, but did not block proliferation of interleukin-2-activated cells. Tetranactin also blocked generation of cytotoxic T lymphocytes and activated killer cells in the mixed lymphocyte culture. However, up to 100 ng/ml tetranactin did not alter the lytic activity of cytotoxic T or NK lymphocytes generated in its absence. The ability of low doses of tetranactin to block the induction of lymphoproliferation is similar to the action of cyclosporin A. Since cyclosporin A is also a cyclic hydrophobic molecule, the immunosuppressive actions of these two agents may involve a similar mechanism.     

Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell

Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) uptake/propidium iodide (PI) exclusion method: above a critical concentration (exposure for 4 h), which was specific for each of the trialkyltin compounds, the cell becomes permeable to PI, indicating loss of viability. Cellular CF fluorescence (derived from intracellular hydrolysis of CFDA) increased as a function of alkyltin concentration below the critical concentration and decreased as viability decreased above the critical concentration. Relative membrane potential, monitored with a cyanine dye (DiOC6), correlated with viability (PI exclusion), remaining essentially unaltered below the critical concentration and decreasing above it. At/above 1 microM TBT, 5 microM TET, or 100 microM TMT, the cell cycle was blocked in the G2/M phase. The 90 degrees light scatter (a measure of refractive index), axial light loss (a measure of volume), and fluorescein isothiocyanate (FITC) fluorescence (a measure of protein content) of nuclei isolated from trialkyltin-treated MELC by detergent treatment, increased as a function of organotin dose. Fluorescence and interference microscopy revealed increased quantities of residual cytoplasmic tags adherent to the nuclei as a function of organotin dose, apparently resulting from increased cytoplasmic resistance to detergent-mediated solubilization. The effects of the trialkyltins correlated with their lipophilicity (octanol/water coefficient). These data support the hypothesis that fixation (protein denaturation, cross-linking, etc.) is an important mode of organotin cytotoxicity.