RESUMO
Objective To study the effect of alpha-melanocyte stimulating hormone (α-MSH) and its novel analogue ( STY39 ) on the production of tissue factor ( TF ) and tissue factor pathway inhibitor (TFPI) stimulated by lipopolysaccharide (LPS) in primary mouse brain microvascular endothelial cells (MBMECs). Methods Female BALB/c mice were selected,purified and primarily cultured for 5 to 7 days. Immunofluorescence assay was use to detect the Ⅷ factor related antigen and identify the MBMEC model. The MBMECs were divided into eight groups:PBS control group, LPS stimulation group, after LPS stimulation 1,2,and 3 h adding 10 -7 mol/Lα-MSH groups or STY39 group (LPS+α-MSH,LPS+STY39) ( n=4 wholes in each group) . The cell culture supernatant and cells were collected at 6 and 8 h after LPS stimulation. An enzyme-linked immunosorbent assay was used to detect the concentrations of TF and TFPI in cell supernatant. RT-PCR was used to detect the expression levels of TF mRNA. Results (1) LPS could induce MBMEC to produce TF and TFPI proteins. The level of TF in the cell culture supernatant reached the peak at 6 h,and the level of TFPI reached the peak at 8 h. (2) At 1,2,and 3 h after LPS stimulating MBMEC,10 -7mol/L α-MSH or STY39 were given. They could significantly decrease the TF protein content in the cell supernatant (P<0. 01),especially the effects of giving α-MSH or STY39 were most significant at 1 h after LPS stimulation (P<0. 05). The effect of STY39 for decreasing TF content was more significant than that of α-MSH (P<0. 05);however,α-MSH and STY39 did not have significant up-regulating effects for LPS inducing MBMEC to produce TFPI. (3) After LPS stimulation,10 -7 mol/Lα-MSH or STY39 were given at different time points. They significantly down-regulated the expression level of MBMEC TF mRNA (P<0. 01). The effect was most significant at 1 h time point (P<0. 05),but there was no significant difference in the effects betweenα-MSH and STY39. Conclusion Bothα-MSH and STY39 can suppress LPS-induced primary MBMEC to produce TF protein and express TF mRNA,and the effect of administration is better after 1 h LPS stimulation. The suppressive effect of STY39 on the production of TF protein is superior toα-MSH.
RESUMO
Objective To evaluate the effect of α-melanocyte stimulating hormone (α-MSH) and its novel analogue STY39 on the production of tissue factor pathway inhibitor (TFPI) in mice with endotoxemia.Methods Female BALB/c mice were randomly divided into eight groups with 9 mice in each group.Endotoxemia was reproduced by intraperitoneal injection of lipopolysaccharide (LPS,25 μg/kg) and D-galactosamine (D-Gal,100 mg/kg).The animals of the control group were given phosphate buffered solution (PBS) instead.In the experimental groups,the mice were injected intraperitoneally with 2.5 mg/kg α-MSH or STY39 at 1,2 or 3 hours following LPS injection.The orbital blood was collected at different time points,and tissues of lung,liver,and kidney were collected 8 hours after the administration of LPS.The plasma TFPI levels were determined by enzyme linked immunosorbent assay (ELISA),and the expression of TFPI mRNA in different tissues was determined with reverse transcription-polymerase chain reaction (RT-PCR).Results The plasma TFPI levels (μg/L) began to increase (11.84 ± 1.55) in the endotoxemia mice 4 hours after LPS challenge and reached the peak (23.49 ± 1.12) at 8 hours.α-MSH or STY39 treatment at 1,2 or 3 hours after LPS challenge could significantly increase the TFPI content,with the best drug effect at 1 hour after LPS challenge (the blood was collected 8 hours after LPS challenge,α-MSH group:58.79 ± 2.67 vs.28.49 ± 1.69,STY39 group:71.08 ± 2.13 vs.28.49 ± 1.69,both P＜0.01),and the effect of STY39 was better than that of α-MSH (P＜0.01).A small amount of TFPI mRNA expression was observed in each tissue of the healthy mice.After LPS challenge,TFPI mRNA expression was increased in all the tissues,especially in the lung,liver and kidney.α-MSH or STY39treatment at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the lung and liver (A value,α-MSH in lung:51.10 ±2.89 vs.32.43 ±2.51,STY39 in lung:72.11 ±3.48 vs.32.43 ±2.51;α-MSH in liver:43.21 ± 2.12 vs.29.29 ± 2.06,STY39 in liver:66.82 ± 1.76 vs.29.29 ± 2.06,both P＜0.01).The treatment with STY39 at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the kidney (A value:45.21 ± 1.80 vs.30.44 ± 2.23,P＜0.01),but the treatment with α-MSH had no obvious effect (A value:24.61 ± 1.98 vs.30.44 ± 2.23,P＞0.05).The enhancing effect of early administration of STY39 on TTPI mRNA expression in the lung,liver and kidney tissues of endotoxemia mice was more powerful than that of α-MSH (all P＜0.01).Conclusion The early administration of α-MSH or STY39 may up-regulate TFPI production in the mice with endotoxemia,and the effect of STY39 is superior to α-MSH.
RESUMO
Objective: To study the expression and activity changes of methionine adenosyltransferase (MAT) in human peripheral T lymphocytes. Methods: The expression of MAT mRNA was detected by RT-PCR and the activity of MAT was measured. Results: After stimulated by IL-2, PHA and anti-CD3 antibody, MAT-Ⅱ gene expression increased by (8.9? 2.1), (7.7?1.9) and (8.0?1.8) times, respectively, and the expression peak was at 8, 4 and 8 h,respectively; MAT activity continuously increased in 48 h. S-adenosylmethioinie (SAM) moderately induced IL-2 and IFN secretion by human T cells. SAM(0.1 mg/ml) downregulated the expression and activity of MAT-Ⅱ and the secretion of IL-2 and IFN induced by PHA or anti-CD3 antibody in human T cells. Conclusion:MAT is involved in the activation of T lymphocytes, and high dose of SAM may also inhibit its activation through PHA and anti-CD3 antibody.
RESUMO
Objective: To investigate the role of T cell in the antitumor immune responses induced by MIF gene-modified tumor vaccine. Methods: MIF gene was transferred into FBL3 erythroleukemia cel l by adenovirus carrier and a new type of tumor vaccine was prepared. The chang es of the number and the function of T cell in spleen and lymph node was observe d. Results: After the mice were immunized with MIF gene-m odified FBL3 vaccine, the number of lymphocyte in spleens and lymph nodes increa sed markedly and the specific CTL activities of splenocytes also increased great ly. FACS analysis showed that the CD3+, CD4+, CD8+ T cells and CD28 posi tive cells in draining lymph nodes of MIF-FBL3 group mice increased more marked ly than that of control groups. When the wild type FBL3 cells were injected into the mice immunized with MIF gene-modified FBL3 vaccine, the growth of tumors w ere obviously inhibited and the survival rate of the mice was increased. Conclusion: It is suggested that MIF gene-modified tumor vaccine can induce specific antitumor immune responses mediated by T cells and may be a candidate for gene therapy of tumor.
RESUMO
Objective:In order to explore the anti inflammatory mechanisms of ? melanocyte stimulating hormone (? MSH), the effects of ? MSH on the production of NO and proinflammatory cytokines in astrocytes induced by LPS were investigated Methods:Rat brain astrocytes cultured in vitro were stimulated with LPS or given ? MSH with LPS stimulation NO produced in astrocytes was tested with Griess reagent IL 1, IL 6 and TNF ? secreted from astrocytes were examined by MTT assay The expression of macrophage migration inhibitory factor (MIF) mRNA was examined with semiquantitative RT PCR analysis Results:The production of NO, IL 1, IL 6, TNF ? and the expression of MIF mRNA were significantly increased in astrocytes stimulated with LPS If giving ? MSH with LPS stimulation, the production of NO, IL 1, IL 6, TNF ? and the expression of MIF mRNA were markedly decreased Conclusion:[WT5”,6BZ]It is suggested that the inhibitory actions of ? MSH on the production of NO and proinflammatory cytokines in astrocytes are related to the inhibitory effects of ? MSH on inflammation in central nervous system
RESUMO
Objective: To investigate the effect of a long-term CpG-ODN stimulation on the maturation of murine bone-marrow derived dendritic cells (BMDCs). Methods: Murine bone-marrow cells were cultured in GM-CSF alone or with CpG-ODN for 7 d or for last 36 h (days 6, 7). Cell phenotypes and antigens uptake by BMDCs were analyzed by flow cytometry. Cytokines released by BMDCs were detected by ELISA. The antigen presenting capability by BMDCs was evaluated by mixed lymphocyte responses.Results:Compared to those of the short-term CpG-ODN stimulation group, the expression of MHCⅡ, CD86, CD40, and secretion of IL-12(p70) by BMDCs in long-term stimulation group were not increased. The phagocytosis of FITC-OVA by BMDCs in long-term CpG-ODN stimulation group was strengthened, but the activation of allogenic and homogenic lymphocyte cells proliferation was impaired. Conclusion:Long-term CpG-ODN stimulation can suppress the maturation of DCs, which may explain the low adaptive immunity in sepsis patients.
RESUMO
Objective: To investigate the mechanisms involved in the induction of antitumor immune responses by MIF gene- modified tumor vaccine. Methods: The changes of the number and the functions of macrophages from the force immunized with MIF gene-modified FBI3 tumor vaccine were analysed.Results: The number,the expression of surface molecules associated with immune responses, the secretion of TNF-?and NO, the phagocytosis, the cytototicity and the antigen presenting activity of peritoneal macrophages from the mice immunized with MIF gene-modified tumor vaccine were all enhanced. Conclusion: The functional enhancement of macrophages is related to MIF secreted by MIF gene-modified tumor vaccine in vivo and the activation of macrophage may play an important role in the antitumor immune responses induced by MIF gene-modified tumor vaccine.
RESUMO
Objective: To study the effects of TCFC on proliferation and osteogenic action of osteoblast in neonatal rat calvaria cultures in vitro. Methods: Osteoblasts were isolated from neonatal rat calvaria through trypsin and collagenase digestion. The proliferation and collagen synthesis were assayed by 3H TdR and 3H proline incorporation. The activity of ALP was measured by p nitrophenyl sodium phosphate assay. The ipriflavone was used as positive control drug.Results: TCFC significantly promoted bone cell proliferation, alkaline phosphatase activity and collage synthesis.Conclusion: TCFC had the anti osteoporosis action by promoting osteoblast proliferation, ALP activity and collage synthesis.
RESUMO
Studies on the immunologic function in patients with Xeroderma Pigmentosum (XP) were reported in this paper. The results showed that the patients had decreased rates of Et-and Ea-rosette formatian of T-lymphocytes;their lymphoblastic transformation ability to PHA were lower than normal individuals; 3 out of 4 patients showed negative OT tests except case 1 was positive; 3 patients gave negative reations to DNCB ( case 3 not tested ); the percentage of ANAE+ cells and SmIg+ cells were in normal range; the number of Ty cells increased in varying degree and most prominent in case 4, while the T? cell did not varied apparently except in case 4 which markedly decreased; the T?/ T? ratios were lower than normal. On the correlation analysis between the percentages of T?, T? or T?/T? ratio and levels of immunoglobulins showed that T? cell was significantly negative correlation with IgG and IgA, while T? cell was very significantly positive correlation with IgA; the T? / T? ratio was significantly positive correlation with IgG and IgA, but they had no correlation with lymphoblastic transformation. Besides T? cell gave significantly negative correlation with T? cell. No autoimmune antibody and circulating immuue complex were detected. It was found that there were aberrations of serum albumin, ?1,?2 and ?-globulins in varying degrees, especially the ?2 macro-globulins (?2M) were markedly increased in all 4 patients by immunoelectraphoretic assay, while ?-globulins were all in normal range. The total complement hemolytic unit (CH50) and C3 content were reduced in case 1 and case 2. The most striking feature was that the ability producing a type interferon (IFN-?) of leukocytes from all 4 XP patients was vary significontly lower than normal controls. Particularlly, bath the titer of IFN-ot produced and 3H-TdR uptake in lymphoblastic transformation of the isolated peripheral blood leukocytes or lymphocytes either prior to or after irradiation with different dosages of ultraviolet rays were all greatly lower in 4 XP patients than normal subjects. The difference of the percentage of IFN-? titer and lymphoblastic transformation rate decreased between XP patients and normal subjects was very sig-nificent. Judging by F test, it showed that there were very significent differences in IFN-? production ability and lymphoblastic transformation rate among different XP patients whose leukocytes or lymphocytes were under UV-irradiation. The decrease of above parameters was more marked in the patient with serious defect of DNA repair function than others, but no difference was seen in normal individuals. These results probably meant that the extent of decreasing IFN-? production and lymphoblastic transformation rate were corresponded with the degree of defect in DNA repair. Discussion on the decrease of immunologic functions seen in the XP patients has been made, which might be one of the important reasons of high cancer incidenc in this disease.
RESUMO
The influences of different concentrations (10-11-105mol/L) ofsubstance P(SP)on the interferon -?(IFN-?) production in mouse spleen cells induced by concanavalin A (ConA) and pokeweed mitogen (PWM) in vitro were studied. The results showed that no influence of 10-11-10-6 mol/L SP on the titre of IFN-? induced by PWM (10?g/ml) was observed; the titres of IFN-? induced by ConA or PWM (5, 2 5 and 1. 25 ?g/ ml) were enhanced by 10-9-10-5 mol/L SP; this enhancement was more significant at 10-7-10-5 mol/L SP. No titre of IFN-? was detected in the presence of SP alone. The titres of IFN-? induced by ConA added at 6h after adding 10-9-10-6 mol/L SP were slightly higher than that induced by ConA and 10-9-10-6 mol/L SP added simultaneously. The titre of IFN-? induced by ConA and 10"5 mol/L SP added in advance tended to reduction rather than enhancement as compared with that induced by ConA and 10-5 mol/L added simultaneously. The effect of enhancing IFN-? production began to appear significantly at lower concentration (10-8 mol/L) of SP added in advance, whereas at 10-7 mol/L SP added simultaneously.