Analysis of cytokines derived from murine yolk sac endothelial cells cultured in vitro

AIM:To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS:The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS:Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor, vWF). The mRNA expressions of cytokines, such as TGF-β2, TNF-α, IFN-γ, FL, BMP-4, MIP-1β, BMP-2A, FLT2, endothelin 2, thymosin β10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION:mYS-EC was purified and expanded in vitro. The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Superantigen SEA antagonizes inhibitory effect of imatinib on T cell activation

AIM：To investigate the effects of staphylococcal enterotoxin A (SEA) on the inhibition of T lymphocyte activation induced by imatinib mesylate (IM). METHODS：Jurkat cells were stimulated with SEA (2 mg/L) and IM (5 nmol/L) for 24 h. The mRNA expression of CD3ε and ζ chains was measured by real-time fluorescence quantitative PCR. The protein levels of CD3ε and ζ chains were detected by Western blotting. RESULTS：The expression of CD3ε and ζ chains at mRNA and protein levels was down-regulated in the Jurkat cells stimulated by IM alone. These down-regulations of CD3ε and ζ chains were reversed by the stimulation of IM combined with SEA. The antagonistic effect of SEA on IM-mediated inhibition of CD3ε mRNA expression was significantly greater than that on CD3ζ mRNA. CONCLUSION:SEA antagonizes imatinib-mediated inhibitory effect on T cell activation.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

沃柑辐射诱变系渝沃无核的果实品质及遗传差异分析

[目的]从果实品质和分子水平上解析造成沃柑辐射诱变系渝沃无核重要表型差异的主要遗传因素,为后续柑橘品种遗传改良奠定基础.[方法]观测渝沃无核的果实品质,应用RNA-seq技术和全基因组重测序揭示渝沃无核的差异表达基因(differential expression genes,DEGs)及基因组的遗传差异.[结果]相比...     

Effects of oxymatrine on lymphocyte proliferation and the quantity of regulatory T cells

AIM: To analyze the effects of oxymatrine (OMT) on the quantity of murine regulatory T cells (Tr cells) in the peripheral blood and mouse lymphocyte proliferation stimulated by Con A, and to probe into the immunological mechanism that OMT treats allergic contact dermatitis (ACD).METHODS: An ACD mouse model stimulated by dinitrofluorobenzene (DNFB) was established. Different dosages of OMT, PBS and hydrocortisone (HCT) were intraperitoneally injected (IP) into the mice. Blood samples were collected at〖JP+2〗 1 d, 7 d, 14 d, 21 d and 28 d, then the T cells were isolated and marked with anti-CD3, anti-CD4, anti-CD25 three-colored immune fluorescence antibody to detect the quantity of CD4+CD25+ T cells with flow cytometry. The fluorescence intensity changes of lymphocytes which were isolated from mouses lymph node and co-stimulated by polyclonal stimulator Con A and OMT were examined by carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining and flow cytometry. RESULTS: OMT at concentrations of 500, 125 and 31 mg/L had the ability to restrain the proliferation of lymphocytes from lymph node in a dose dependent manner. However, OMT at concentrations of 16, 8, 4 and 2 mg/L promoted the proliferation of T lymphocytes from lymph node, but was not obviously dependent on its concentration. Intraperitoneal injection of OMT increased the numbers of CD4+CD25+T cell in peripheral blood obviously (P<0.01). CONCLUSION: The effects of OMT on the proliferation of T lymphocytes from mouses lymph node cells are observed, OMT also increases the CD4+CD25+T cells in the peripheral blood, implying that OMT is a kind of immunoregulator with dual effects.     

Recent advances in the correlations between dendritic cells and regulatory T cells

Dendritic cells (DCs), representing a heterogenous population of professional antigen-presenting cells, are the initiators and modulators of the immune responses. Studies indicate that regulatory T cells contribute to immune nullipotency and immune suppression via cell-cell contact or cytokine secretion. These two kinds of cells may be valuable tools for modulating immunity in the setting of auto-immunity, cancer, chronic viral infections and graft rejection, etc. Here we discuss the current knowledge on the functions of regulatory T cells and denditic cells-based immunoregulation and the applications.     

Expression and regulatory effect of P73 antisense RNA 1T in tumors

The discovery of abundant new long non-coding RNAs (lncRNAs) and extensive investigation of their roles in various diseases have been reported, especially in cancers. The lncRNA P73 antisense RNA 1T (TP73-AS1) is involved in dysregulation of cell signaling and closely correlated with cancer development, progression, and response to therapy. This review is a brief update of the current knowledge related to the role of TP73-AS1 in cancer-associated molecular pathways and pathophysiology, and possible determinants for TP73-AS1 to function as a biomarker, aiming to stimulate the basic investigation of TP73-AS1 as well as its translation to clinical applications.     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

Foxp3-transduced polyclonal regulatory T cells suppress cytotoxicity of NK cells

AIM: To investigate the effect and mechanism of Foxp3-transduced CD4+CD25-T cells on the cytotoxicity of NK cells. METHODS: Retroviral Foxp3 gene transfection was applied to nave CD4+CD25-T cells. Fresh transduced CD4+Foxp3+ T cells were co-cultured with NK cells. ［51Cr］ labeled YAC-1 cells were used to detect NK cells cytotoxicity. The anti-TGF-β antibody was added into the co-culture system to detect the TGF-β blocking effect. Also the transwell co-culture system was used to investigate the regulatory effect of Treg cells on NK cells. RESULTS: One week after transduction, 38.0% of Foxp3-transduced T cells showed GFP expression by flow cytometry. Foxp3-transduced CD4+CD25-T cells suppressed function of NK cells. The inhibition rates of Foxp3 transduced CD4+CD25- T cells were 42.9% at 24 h and 22.7% at 48 h. When anti-TGF-β antibody was added to the co-culture system, the inhibition rate of CD4+Foxp3+ T cells was 3.2% and 2.1%, respectively. CONCLUSION: CD4+Foxp3+ T cells significantly inhibit the cytolytic function of NK cells. TGF-β plays different roles on this action in different inhibition systems. The inhibitory effect of Treg cells on NK cells is cell-to-cell contact dependent and associates with TGF-β expression.     

Effect of estrogen on murine vaginal epithelial cell anti-Candida activity

AIM: To investigate the growth inhibition of Candida albicans mediated by vaginal epithelial cells and determine if estrogen affects the anti-fungal activity. METHODS: C57BL/6J mice vaginae from estrogen treatment group, diestrus group and ovariectomized group were excised, respectively. The vaginae were dissociated into single cell suspension by dispase and collagenaseⅠ. The epithelial nenriched cells were used as effector cells. Blastoconidia of C. albicans were used as target cells. After coincubation of effector with target for 9 h, the target cells growth density was observed, percent growth inhibition was calculated, and ultra-structural changes were observed. RESULTS: After coincubation of effector cells with target cells for 9 h, growth density of C. albicans was visibly reduced and it's growth activity was inhibited. Compared to ovariectomized group and diestrus group, vaginal epithelial cells from estrogen-treated mice had less ability to inhibit the growth of C. albicans (P<0.05). C. albicans incubated alone showed intact and legible ultrastructure while the Candida coincubated near the epithelial cells showed obvious changes: the cell wall ruptured, intact cytoplasmic membrane was damaged, intracellar component dissolved. CONCLUSION: The results suggest that mice vaginal epithelial cells have innate anti-Candida activity in vitro and the activity was inhibited by estrogen.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Studying the spatial evolutionary behavior of urban forest patches from the perspective of pattern-process relationships

Urban forest dynamics can influence the provision of ecosystem services provision. Considerable research has been conducted to understand how these dynamics respond to urbanization, from individual patches to entire landscapes. However, most of these are cross-sectional studies based on landscape metrics, and research using a process-based perspective in this context is scarce. In this study, we present a “pattern-process” analytical framework to quantify the evolutionary behavior of urban forest patches. We combine this framework with land cover classification data based on high-resolution remote sensing images (< 1 m) from 2002, 2013, and 2019 to detect the dynamic characteristics of four processes of forest patches in Beijing urban areas. These dynamic characteristics include: size distribution, aggregation and fragmentation, transfer, and self-stabilization. The results showed that 1) the average size of the patches in the study area is increasing, and patches larger than 50 m² have a more positive influence on the process of patch structure evolution, 2) patch fragmentation shifts with the direction of urban sprawl, 3) transfer between urban forest and bare land is increasing, and 4) urban forest network construction positively enhances the stability of patches. This framework can provide a useful basis for understanding the spatial and temporal evolution of urban forest landscapes during urban development and contribute to the sustainable management of urban forests.     

Dual immunoregulatory effects of CD69 on cell activation and apoptosis

CD69, a member of C type lectin superfamily and NK cell gene family, plays important roles in signal transduction in the regulation of cellular functions. Previous studies demonstrate that CD69 has a vital role in cell activation and CD69 is used as a marker of cell activation because of its early expression in activated cells. CD69 is also the costimulatory signal for cell activation, proliferation and differentiation. Furthermore, recent studies show the close association between CD69 and apoptosis. CD69 mediates apoptosis selectively in activated cells, mainly in activated eosinophils and monocytes. Using CD69 as an apoptotic inducer in certain cells may have good result and less side effect. This article tries provide a brief summary of the dual immunoregulatory effects of CD69 on cell activation and apoptosis.     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

Implantation study on the potential of murine epidermal stem cell differentiation

AIM: To investigate the potential of murine epidermal stem cell (ESC) differentiation after seeded in a biodegradable carrier and implanted subcutaneously into syngeneic recipient mice. METHODS: ES cells were induced in vitro to differentiate into ESCs. After stained with a fluorescent dye Hoechst 33342, these ESCs were seeded into a polyglycolic acid (PGA) net containing collagen gel, functioning as a cell carrier, and implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells. RESULTS: The ESCs kept alive in the implant when observed under a fluorescent microscopy 3 weeks or longer after implantation, and could differentiate into hair follicle-like structure, glandular structure, and gave rise to additional structures displaying features resembling native dermis. No apparent rejection or severe side effects were observed at least 10 weeks post-implantation. CONCLUSION: It is feasible to use these ESCs as seed cells in the study to fabricate dermal equivalent having the potential to develop dermal appendages.     

Effects of inactivated HIV-1 particles on human CD4+T cell activation and Th1/Th2 cytokine secretion in whole blood

AIM: To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 μg/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62±0.63) %, whereas it was (38.82±6.00)%, (3.83±1.07)%, (5.94±0.85)% and (9.30±1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-α and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.