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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 ?mol/L and 50 ?mol/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 ?mol/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 G_0/G_1 phase to S and G_2/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.
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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 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
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Objective To investigate the status of cellular immunity from Th cell polarization in pa-tients with different courses of condyloma acuminatum(CA).Methods The isolated PBMC were polarized by PHA and self-plasma for72hours,then followed by two-color immunofluorescent staining with anti-CD4-PE and anti-CCR5-FITC,or with anti-CD4-FITC and anti-CCR3-PE.Finally the stained cells were analyzed by flow-cytometry.Results The percentages of Th1/Th2cells of the short-course group and long-course group were(25.82?2.22)%/(14.80?1.14)%and(12.20?1.37)%/(13.74?0.99)%,respectively;the differ-ences between normal control and two CA groups were significant(P
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AIM: To investigate the inhibitory effects of genistein(Gen) on mouse allergic contact dermatitis(ACD).METHODS: The animal model of ACD was induced by DNFB.The effects of different doses of Gen on mouse ear swelling,body weight,histopathological changes in mouse ear skin,thymus index and spleen index were observed.RESULTS: All groups of Gen inhibited mouse ear swelling induced by DNFB significantly.The infiltration of inflammatory cells and thymus index were also reduced.However,the increase in mouse body weight was not affected.Low dose of Gen increased spleen index,high dose of Gen decreased spleen index.CONCLUSION: Genistein has significant inhibitory effects on mouse ACD induced by DNFB.
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AIM: To explore the inhibitory effect of oxymatrine (OMT) on the allergic contact dermatitis (ACD) stimulated by dinitrofluorobenzene (DNFB) and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT, PBS and hydrocortisone (HCT) respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.
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AIM: To construct the mammalian cell expression vector for enhanced green fluorescent protein (EGFP) and HLA-A*0201 fusion protein and analyze its expression and subcellular localization in the transfected K562 cells. METHODS: The HLA-A*0201 cDNA was cloned by RT-PCR and the gene was inserted into pEGFP-N1 to construct a vector for the fusion protein. The expression of the fusion protein in K562 cells transfected with the vector was evaluated by flow cytometry and its subcellular localization was investigated by confocal microscopy. RESULTS: The full-length encoding region of HLA-A*0201 cDNA was cloned from two HLA-A2 positive donors and the expression vector for the HLA-A*0201-EGFP fusion protein was constructed by PCR using a primer pair to introduce a Kozak sequence before ATG and the stop codon was deleted. Five hours after K562 cells was transfected with the vector, the expression percentages of HLA-A*0201 and EGFP were 25.12?2.26 and 27.37?3.59, respectively and no significant increase was observed after 24 h. The fusion protein was predominantly located on the membrane with low level distribution within the cells. In contrast, no HLA-A*0201 but only EGFP was detected in the empty vector transfected K562 cells and the EGFP was dispersed within the cells. CONCLUSIONS: The expression vector for HLA-A*0201-EGFP fusion protein was constructed and the fusion protein expressed in K562 cells was primarily distributed on the membrane. The results suggest that the transfected K562 cells are potential antigen-presenting cells.
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AIM: To study the suppressive effect of glycyrrhizin (GL), a Chinese medicine, on DNFB-induced contact hypersensitivity in mice. METHODS: BALB/c mice were divided into 5 groups according to different medication: GL 1 (11 mg/kg) group; GL 2 (22 mg/kg) group; GL 3 (44 mg/kg) group; dexamethasone (DXM, 0.75 mg/kg) group; and normal saline group. For induction of contact hypersensitivity (CHS) to DNFB, mice were sensitized to abdomen and challenged to right ear by epicutaneously DNFB. Each mouse was administrated intraperitoneally on day 1 to day 5 with different medication. The suppression of mice CHS by different medication were evaluated 24 hours after elicited, according to ear thickness difference, ear weight difference and pathological change of challenged ear section. Thymus index and spleen index were calculated to see the effect on mouse immune system to CHS. RESULTS: Compared with normal saline group, the ear thickness difference and ear weight difference were both significantly reduced in GL1, GL2, GL3 and DXM groups (P
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AIM: To study the suppressive effect of c hu ankezhi (CKZ) injection, a Chinese medicine, on murine allergic contact dermatit is (type IV hypersensitivity). METHODS: Mice were divided into 6 groups according to different medicine treatments: CKZ high, middle, low dose ( CKZⅠ,Ⅱ,Ⅲ) groups, dexamethasone(DEX), benadryl and saline groups. Murine alle rgic contact dermatitis was induced by intraperitoneal injection of 2, 4-dinitro fluorobenzene. Different medicines were administrated at 2 h before sensitizatio n on day 0 and day 1, day 2, 2 h before elicitation and 6 h after on day 5. The six experimental groups were compared according to left ear thickness difference (S1), left ear weight difference (S2), body weight difference (S3) and dermal i nflammatory infiltration cell number. RESULTS: Compared with saline group, the left ear swelling and d ermal inflammatory infiltration cell number were significantly reduced in CKZⅠ, Ⅱ,Ⅲ and DEX groups (P
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AIM: The expression of CD28, CD56 and CD57 on CD8+T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS: Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8+CD28+, CD8+CD56+ and CD8+CD57+T cells in the peripheral blood between the young and elderly groups.RESULTS: CD8+CD28+T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07?5.28 and 49.84?7.43,respectively (P
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AIM: To investigate the details of CD4 + T cell polarized to Th1/Th2 in vitro . METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro , these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4 +T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro . The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%?1.59%)/(12.70%?1.65%),(17.19%?1.03%)/(17.50%?1.30%),(19.49%?2.87%)/(18.58%?1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4 +T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro , and the cell number of Th1 and Th2 would be in balance.