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Astragalus membranaceus (A. membranaceus) is a widely used traditional herb in China and Korea. A. membranaceus polysaccharides (AMP), which make up a major part of the root extract, have been shown to modulate immune modulations, especially activation of bone marrow-derived dendritic cells (BMDCs) and T cells. However, the immune stimulatory effect of AMP in the mouse in vivo and human peripheral blood DCs (PBDCs) has not been well investigated. In this study, we found that intravenous (i.v.) injection of AMP in C57BL/6 mice induced remarkable elevations in co-stimulatory and MHC class I and II molecule levels in the splenic DCs and its subsets. The stimulatory effect of DCs by AMP was elevated 6 h after treatment, which rapidly decreased 18 h after injection. Furthermore, AMP promoted intracellular production of pro-inflammatory cytokines in spleen DC subsets, which contributed elevation of serum cytokine levels. Finally, the AMP promoted PBDC activation. Thus, these results demonstrate that AMP can be used as an immune stimulatory molecules in human and mouse.
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Objective To investigate the regulating function of human gingival mesenchymal stem cell (GMSC) on the proliferation and differentiation of B cells and its underlying molecular mechanism. Methods GMSC were isolated and B cells were isolated from peripheral blood. GMSC or fibroblasts were co-cultured with B cells in vitro and assigned into the GMSC group and fibroblast group. The proliferation of B cells was detected in two groups. The expression of IgG1 and IgM in the cell supernatants was measured between two groups. The secretion of interleukin (IL)-6, Perforin, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was compared between two groups. The expression levels ofIL-10 and transforming growth factor (TGF)-β in B cells were detected between two groups. The expression of PC-1 in B cells was measured in two groups. The signaling pathway involved with the regulating effect of GMSC on B cell function was investigated. The regulating effect of GMSC on the role of B cells in activating T cell function was assessed. Results Compared with the fibroblast group, the proliferation of B cells was significantly weakened in the GMSC group (P < 0.05). Co-culture of GMSC and B cells significantly inhibited the secretion of IgG1 and IgM from B cells and the secretion ofIL-6, Perforin, IFN-γ and TNF-α (all P < 0.05). Compared with the fibroblast group, the secretion of IL-10 and TGF-βwas significantly higher in the GMSC group (both P < 0.05). The expression level of PC-1 in the GMSC group was significantly down-regulated (P < 0.05). After adding ALK5, an inhibitor of TGF-β receptor, the inhibitory effect of GMSC upon B cells was significantly weakened (P < 0.05). Compared with the fibroblast group, the ability of B cells to activate and proliferate T cells was significantly attenuated in the GMSC group (P < 0.05). Conclusions GMSC can inhibit B cells and their mediated immune responses. The activation of B cells and other related functions can be suppressed through the TGF-β signaling pathway.
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Objective To explore the effects of surface functionalized multi-walled carbon nanotubes (FMWCNTs) on the cytotoxicity of human peripheral blood mononuclear cell (PBMC).Methods Five different types of MWCNTs (hydroxylated,carboxylated,aminated,nickel-plated and pristine MWCNTs (P-MWCNTs)) with the same diameter and length were evaluated the dispersion and characterizations in physiological salt solution by transmission electron microscopy.PBMC were isolated by density gradient centrifugation from human peripheral blood,and 5 types of MWCNTs were ultrasonically dispersed in serum-containing medium respectively.After incubation with PBMC for 12,24,48 or 72 h,cytotoxicity was detected by CCK-8 kits.Results All the MWCNTs had well dispersion,especially the F-MWCNTs.Cytotoxicity results showed that all types of MWCNTs could induced PBMC death,and presented dose-dependence manner and a certain degree of time-dependence manner.Compared with the P-MWCNTs,F-MWCNTs changed cytotoxicity statistically,with the hydroxylated,carboxylated,aminated MWCNTs weakened,aminated MWCNTs significant (P＜0.05),nevertheless the nickel-plated MWCNTs increased.Compared with the P-MWCNTs (25 μg/ml),cell viability of PBMC after 24 and 48 h incubation with the same dose of nickelplated MWCNTs both decreased,and the differences was statistically significant (P＜0.01,P＜0.05).Conclusions The functional group modification affects not only the MWCNTs dispersion in medium,but also the cytotoxicity of the MWCNTs on PBMC.
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Objective To study the effect of γ-rays irradiation on the differentiation potential of the human peripheral blood monocytes (PBMCs) into osteoclast-like cells (OCLs) in vitro.Methods PBMCs were isolated by density gradient centrifugation,treated by receptor activator of nuclear factor-κ B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and exposed to 137Cs γ-rays with different radiation doses (0,0.75,2 Gy).After seven days of incubation,the cells were stained for tartrate resistant acid phosphatase (TRAP) and bone slices were stained by toluidine blue on the tenth day.Meanwhile,the characteristic osteoclast markers including Cathepsin K and integrin β3 were analyzed by real-time PCR.Tartrate resistant acid phosphatase 5b (TRAcP-5b) in the culture supernatant wasdetermined by ELISA.Results PBMCs were differentiated into OCLs by the treatments of RANKL and MCSF.The number of TRAP positive multinucleated OCLs was significantly higher in the dose of 0.75 Gy group than in control (0 Gy) group (t =3.451,P ＜ 0.05).Compared with the control group,the expression levels of Cathepsin K and integrin β3 and the concentration of TRAcP-5b were significantly elevated (t =2.343,2.728,3.631,P ＜ 0.05).However,in the 2 Gy group,there was a decrease in the number of osteoclasts,mRNA expression level of osteoclast characteristic markers and TRAcP-Sb,but no statistically significant differences compared with the control group.Conclusions Ionizing radiation may influence the osteoclastogenesis during the PBMCs differentiation to OCLs.At low dosage,ionizing radiation promotes osteoclastogenesis and enhances the resorptive activity of osteoclasts,but a decline of differentiation potential was observed at high dosage of radiation.
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ObjectiveTo induce human peripheral blood mononuclear cells differentiate into hepatocyte-like cells by hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in vitro and determine whether PKH26 could be used to serve as an effective tracer for the cells,and observe the ability of transplanted hepatocyte-like cells differentiate into hepatic cells in nude mice.MethodsGroup A and B were set up respectively.In Group A,mononuclear cells were cultivated without hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in cell culture.They were used as negative control group.In Group B,mononuclear cells were cultured with the administration of both HGF and FGF-4 to induce the differentiation into liver hepatocyte-like cells.The changes in cell morphology were observed and the expressions of AFP and CK 19 were detected by immunocytochemical staining in two groups at different times after induction.The hepatocyte-like cells differentiated from human peripheral blood mononuclear cells labeled by the fluorescent dye PKH26 injected into caudal vein in nude mice is experimental group.The nude mice injected with equal amount of normal saline in control group.The migration of the labeled cells into the liver are observed by the fluorescence microscope in the hepatic tissue sections of nude mice and the expressions of ALB were detected by immunocytochemical staining two weeks after the cells transplantation.ResultsCells in group B have a strong proliferative activity.It becomes large and oval,grows in colonies following induction.Cells in group A that showed spherical shape when peripheral blood mononuclear cells were just isolated are gradually becoming inconformity in morphology,spindle or fibroid,and a few cells are round:cells developed apoptosis and cracked following incubation.The expressions of AFP and CK19 were positive after induction in group B as detected by immunocytochemicat staining.Inversely,the expressions of AFP and CK19 were negative in group A after incubation.The experimental group showed numerous PKH26 labeled cells in the hepatic tissue sections of nude mice.But the control group did not show PKH26 labeled cells.The expressions of ALB were positive in the experimental group as detected by immunocytochemical staining after two weeks of the cells transplantation.ConclusionHuman peripheral blood mononuclear cells have the potential to differentiate into hepatocyte-like cells under the induction of HGF and FGF-4.Additionally,PKH26 is an effective tracer in hepatocyte-like cell transplantation.The hepatocyte-like cells settled in hepatic tissue begin to differentiate into mature hepatocyte after two weeks of the cells transplantation.It plays hepatic cells function and expresses alhnmin.
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Objective To investigate the effects of 60Co γ-ray partial radiation on chromosome aberration in human peripheral blood in vitro.Methods The samples of heparinized peripheral whole blood from 3 healthy persons were exposed to 60Co γ-rays at the doses between 0 and 8 Gy with the dose rate of 0.35 Gy/min at the temperature of 37 ℃ ,and then mixed with the unirradiated blood samples of the Microscopy was used to observe the chromosome aberration double ( centromere + centromere) and the biological dose was estimated thereby.ResultsThe amounts of double centromere + centromere were increased along with the dose of irradiation in all groups.The estimated biological dose was higher than the 1/3 of the irradiation dose when the dose was between 0.5 to 2 Gy,and was close to the 1/3 of the irradiation dose when the dose was between 4 to 8 Gy.Conclusion Chromosome aberration can be used as a biomarker in estimation of uneven irradiation.
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Objective To detect the expression of DNA damage response genes induced by radiation in human peripheral blood lymphocyte,and to explore the new biomarkers of radiation.Methods The human peripheral blood cells were irradiated to X-rays at different doses of 0,1,2,3,4,and 5 Gy.The quantitative real.time qPCR wag used to detect the expressions of cyclin-dependent kinase inhibitor l a gene(Cdknl a)and growth arrest and DNA damage inducible gene(Gadd45a)in lymphoeytes at 4 and 24 h post-irradiation,respectively.The method of CB mieronucleus was used to determine the change of micronucleus ratio.Results The expression of Cdknl a in peripheral blood lymphocytes wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy.reached the peak at 4 Gy and began to decrease at 5 Gy,which showed a dose-dependent manner(r=0.946,0.975,P<0.05).Similarly,the expression of Gadd45α in human peripheral blood lymphocytes was also increased significantly at 4 and 24 h post-irradiation to 0-5 Gy in a dose-dependent manner,while the expression of Gadd45a at 4 h wag higher than that at 24 h(r=0.936,0.797,P<0.05).The ratio of micronuclei wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy(r=0.990,0.984,P<0.05).Conciusions Cdknl a and Gadd45α expression could be increaged significandy at 4 and 24 h post-irradiation to 0-5 Gy,showing a good linear relationship.which might be candidate for radiation biological dosimeter.
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Objective To analyze the chromosome aberrations induced by partial radiation in human peripheral blood lymphocytes in vitro.Methods Heparinized whole blood samples were exposed to 2 Gy ~(60)Co 7-rays at 37℃ ,and then mixed with non-irradiated blood by different ratio.The slides were prepared after culturing and the unstable aberrations were analyzed.Results The chromosome aberrations had a good relationship with the ratio of irradiated blood.The chromosome aberrations in partial irradiated group were higher than that in the irradiated group.The estimated dose was 1.27 Gy when the ratio was 1 : 1 ,greater than the dose of 1 Gy.The estimated dose was 0.93 Gy when the ratio was 0.5＝1,also greater than 0.5 Gy.But when the ratio was 1:0,the radiation dose was accordant with the estimated dose.Conclusions Chromosome aberrations could be a biomarker for estimating the uneven irradiation.
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The gene expressions of Interleukin-6 (IL-6) from human peripheral blood lymphocytes (HPBL) stimulated by C. albicans were investigated by using ELISA (Enzyme linked immunosorbent assay), reverse transcription polymerase chain reaction (RT-PCR) and northern blotting. HPBL (1 X 10'/ml) obtained from normal human peripheral blood lymphocytes were cultured with live C. albicans (LCA) or heat killed C. albicans type A 311 (KCA, 3 X 10/ml) for various times (0.5, 1, 4, 8, 18, 24, 48 and 72 hours). On the purpose of this experiment, we also used lipopolysacchalide (LPS, 10 ug/ml), zymosan (1, 10, 100 ug/ml) as a polysacchaide component of the wall of yeast cells or TNFa (50, 100 ng/ml) as a IL-6 inducers. For observation of the level of IL-6 gene expression, actinomycin D (AD, 5 pg/ml) or cyclohexamide (CHX, 25 ug/ml) was added to HPBL stimulated with LCA for 0.5, 2, 4 hours and the HPBL were assessed for IL-6 mRNA. The highest value for IL-6 activity by LCA were observed at 48 hours reaction, but in the case of KCA, highest value of IL-6 activity was observed at 72 hours reaction and the value was also higher (500 pg/ml) than that of LCA (188 pg/ml). 1L-6 mRNA induced by LCA were detected up to 48 hours but in the case of KCA, the band for IL-6 mRNA were far stronger and appeared until lately than that of LCA. Therefore, the results of IL-6 gene expression agreed with that of ELISA.
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Humans , Blotting, Northern , Dactinomycin , Enzyme-Linked Immunosorbent Assay , Gene Expression , Hot Temperature , Interleukin-6 , Lymphocytes , Polymerase Chain Reaction , Reverse Transcription , RNA, Messenger , Yeasts , ZymosanABSTRACT
0.05), but were highly significant at doses of 100 ?mol/L or above (P
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Objective:To study the regulation effect of water extraction from Ganoderma Lucidum (Lyess. ex Fr) karst (GL W) on human immune cells and cytokine. Methods: With the technologies of MTT and FACS, the proliferation of T lymphocyte subgroup and IL 6 production in human peripheral blood lymphocytes (PBL) were studied. Results: 1. In the absence of mitogin, GL W could induce not only the inactive lymphocyte but also the PHA activited lymphocyte to proliferate in the similar concentration. The proliferation in the later was more obvious ( P
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The RNA dot hybridization with human IL-6 cDNA showed that panaxatriol saponin (PTS) enhanced the interleukine-6(IL-6) mRNA concentration within PHA activated peripheral blood mononuclear cell (PBMC) byabout 4 folds,suggesting that PTS could promote the IL-6 gene transcription of lymphoid tissue
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Lymphocyte chemiluminescence (Ly-CL) is one of the early events involved in the activation of lymphocytes. This CL is thought to be result ed from the reactive oxygen species (O_2, H_2 O_2, OH, 'O_2) produced by the lymphocytes, the consequent oxidation of luminol and emission of light. In this paper we investigated the experimental condition for ConA-induced human peripheral blood lymphocyte (PBL) CL by using orthogonal design. The effects of new born calf serum (NCS), bovine serum albumin (BSA), temperature and time of cell storage on CL were also studied. The results show that the optimal condition of Ly-CL assay was 1.0 ml PBL suspension (1.4 ? 10~6 cell/ml ), 0.2 ml Luminol solution ( 7 ? 10~(-4)M) and 0.2 ml ConA ( 700 ?g /ml ). The isolated PBL were suspended in phenol red-free Hanks solution containing 0.1% BSA and can be kept for 2 hr at 4℃ before being used without adversely affecting cell viability and CL.
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With use of ~(125)I-udR release test,a significant natural killer activityagainst K_(562) target cells has been determined in human peripheral wholeblood(WB).r There was a gradual decrease in NK activity as rise inthe dilution or WB.In comparison with peripheral monouclear cells(PBMC)oneself,the NK activity lowered distinctly at both 1:4 and 1:8 dilution ofWB(p0.4).It seems that neither the red blood cells nor thePlasma ffeect on NK activity of WB,The NK activity in WB as well as inPBMO was improved by incubation with human leukocyte interferon.TheNK activity of WB and PBMC in patients with hepatocellular carcinomawas signiticantly lower than in normal controls.The results showed tahtNK activity may be determined by using properly diluted WB instead orPBMC,and the assay can be easily performed in clinical practice.
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Objective: To explore the effects of Lycium barbarum polysaccharide (LBP-X) on IL-2 and TNF-? gene expressions in human peripheral blood mononuclear cells. Methods: RT-PCR (reverse transcription-polymerase chain reaction) was used to detect the gene expressions of IL-2 and TNF-?. Results: LBP-X at 5-40 mg/L upregulated the levels of IL-2 and TNF-? mRNA in human peripheral blood mononuclear cells after optimal time culture. Conclusion: The immunoenhancing effect of Lycium barbarum polysaccharide may be associated with its stimulation of IL-2 and TNF-? gene expressions.
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Objective To establish an effective method of isolating and culturing circulating fibrocytes from human peripheral blood and study the relationship between the expression specific molecule markers expression and the morphological characteristics. Methods Total peripheral blood leukocytes were isolated from human peripheral blood by being centrifuged over Ficoll-Paque and cultured in DMEM supplemented with 20% fetal calf serum.Adhered cells were detected with immunocytochemistry,FCM and electron microscopy.The collagen synthesis was studied by measurement of the hydroxyproline concentration in the medium with chemical method. Results After 9 days cultue in vitro,CD34,CD45 and collagen Ⅰ staining was positive and 83.5% of these cells secreted collagen Ⅰ detected by FCM.Electron microscopy of circulating fibrocytes showed morphological characteristics of fibroblasts.The hydroxyproline concentration in the medium was 11.17%mg/L,which was statistically and significantly different compared with 8.07mg/L in the control medium(P