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Objective To explore the effects of vasoactive intestinal peptide (VIP)on the ratio of CD4+CD25+Treg/CD4+T cell and the expression of TGF-β1 in experimental autoimmune encephalomyelitis (EAE)rats. Methods We randomly divided 60 healthy female Wistar rats into normal control group,EAE control group,VIP low-dose group and VIP high-dose group.We used myelin basic protein (MBP)+ complete adjuvant (CFA)to establish EAE model. Since the day of model construction, the low- and high-dose VIP groups received intraperitoneal injection of 4 nmol/kg (0.2 mL)and 16 nmol/kg (0.8 mL)of VIP every other day,respectively;normal control group and EAE group received injection of saline of 0.8 mL for 10 days in a row.We recorded the peak of neurological dysfunction score (NDS)changes in the rats,observed the pathological changes and GFAP+astrocyte activation in the brain at the morbidity peak of rats with HE staining,and detected the ratio of CD4+CD25+Treg/CD4+T in the spleen with FACS and TGF-β1 cytokine level in brain tissue with ELISA.Results The peak nerve dysfunction score was decreased in each VIP dose group.In normal control group,there were decreased inflammatory cell infiltration and decreased number of active astrocytes in the brain tissue.The degree of infiltration of inflammatory cells and astrocyte activation in VIP control groups were significantly lower than those in EAE group.The CD4+CD25+Treg/CD4+T cell ratio of the spleen tissue in each dose VIP treated group rats was higher than that in EAE control group.The cytokine level of TGF-β1 in the brain tissue increased in each VIP dose group in the dose-dependent manner.Conclusion Through up-regulating the ratio of CD4+CD25+Treg/CD4+T cell in the spleen tissue,increasing TGF-β1 content in brain tissue,and inhibiting the infiltration of inflammatory cells and the astrocyte activation,VIP plays an important role in prevention and control of EAE.
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@#AIM: To investigate the role of regulatory T cells in the pathogenesis of autoimmune dry eye and to analyze the expression of endogenous regulatory T cells and related cytokines in the lacrimal system to provide a more effective treatment for autoimmune dry eye disease treatment programs. <p>METHODS: Rabbit lacrimal gland epithelial cells isolated and cultured for a period of time were separately cultured and mixed with isolated peripheral blood lymphocytes in a ratio of 1: 1 and cultured with 5-Bromo-2-deoxyUridine(BrdU)to detect the proliferation of peripheral blood lymphocytes, and the activated autologous peripheral blood lymphocytes were transferred to donor rabbits via ear vein. The rabbits were used as the rabbit model of autoimmune dry eye and the normal rabbits which did not induce the disease as the control group. Before and after the experiment, tear film rupture time and tear secretion at 2,4,6,8wk,were observed. At 4 wk after the transfusion the rabbits were sacrificed for the collection of bilateral upper and lower lacrimal gland and conjunctiva, and pathological HE staining, and then extract the total RNA in lacrimal gland. The expression of IL-17, TNF-α, IL-6 and TGF-γ was detected. <p>RESULTS: BrdU detected peripheral lymphocyte proliferation rate of 3.72. Compared with the normal group, the time of rupture of the tear film decreased(<i>P</i><0.05), and the tear of the model group was significantly lower than that of the control group(<i>P</i><0.05). The expression of IL-17, TNF-α, TGF-γ and IL-6 in the model group were significantly higher than those in the control group(<i>P</i><0.05). The expression of IL-10 and TGF-β was significantly lower than those of the control group(<i>P</i><0.05). The ratio of CD4<sup>+</sup> CD2<sup>+</sup> Treg cells in lacrimal gland tissue and spleen was higher in control group. The proportion of CD4<sup>+</sup>CD25<sup>+</sup> Treg cells in model group decreased(<i>P</i><0.05). <p>CONCLUSION: IL-17, TNF-α, IL-6 and TGF-γ play important roles in inflammatory reaction of lacrimal gland and the immunodepression active by CD4<sup>+</sup>CD25<sup>+</sup>Treg cells decreases when the rabbit model of dry eye is involved.
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Objective To study the phenotype and function of CD4 +CD25 + Treg cells in the peripheral blood of patients with systemic sclerosis (SSc) and their relationship with fibrosis.Methods The proportion of Foxp3, CD127, CTLA-4 and CD69 on CD4+CD25+ Treg cells in peripheral blood were detect by flow cytometry;the levels of TGF-[1 and IL-10 in serum were detect by enzyme-linked immunosorbent assay (ELISA) in patients with SSc.The correlation between Treg cells and the score of chest HRCT, MRSS, and disease activity was analyzed.T test and Pearson correlation analysis were used for statistical analysis.Results ① Compare to the control group, the proportion of CD4+CD25+ Treg cells in peripheral blood of SSc patients was increased significantly(12.9±2.4 vs 14.9±2.2, t=2.63, P=-0.012), and the expression of CD69+, CTLA-4+ on CD4+CD25+ Treg cells was decreased significantly (P＜0.01).② Compare to the control group, the proportion of CD4+CD25+Foxp3 + cells and CD4+CD25+CDI27-cells in peripheral blood of SSc patients was increased significantly (respectively, 3.3±0.7 vs 5.0±0.7, 5.1±1.6 vs 7.6±2.0, t=7.03, 4.195;P＜0.01), but no correlation between them was detected.③ The level of TGF-β1 in the serum of the SSc patients was lower than that of the control group(86±29 vs 133±29 ng/ml, t=-5.026, P=0.000).However, IL-10 had no significant difference between the two groups.④ The proportion of CD4+CD25 +Foxp3 + cells and CD4~D25 +CD127-cells in peripheral blood of SSc patients was positively correlated with the scores of chest HRCT (respectively, r=-0.541, P=0.02;r=0.486, P=0.041), and no correlation was observed with ESR, CRP.In addition, CD4+CD25+Foxp3+ cells were associated with MRSS.Conclusion The proportion of CD4+CD25+ Treg cells in the peripheral blood of SSc patients is increased, but they alters the immune function.The different phenotypes of Treg cells of CD4+CD25+Foxp3+ cells and CD4+CD25+CD127-cells in peripheral blood of SSc patients are increased significantly, which changes along with skin and lung fibrosis.The associated cytokine TGF-β1 is reduced, and IL-10 is not significantly changed.
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Objective To observe the effects of simvastatin combined with aspirin on the heart allograft and detect its mechanism. Methods Heterotopic heart transplantation was performed from Wistar to Sprague-Dawley (SD) rats. All SD rats were randomly assigned to Sham; HT (heart transplantation); HT + simvastatin(HT + S);HT + aspirin (HT + A); HT + aspirin + simvastatin (HT + A + S) group at different time points (day 3, 7, 10, 15, 20, 30 and 40) after transplantation (n=20). eNOS expression was detected by immunohistochemical methods and NO levels was measured by Griess assay. Meanwhile , the analysis of CD4+CD25+Tregs was performed by flow cytometry and histological examination for pathological change of heart and vascular. Results Simvastatin combined with aspirin significantly prolonged the mean survival time of heart allografts in HT + A + S group to (39 ± 5.3) days (n = 19) and in HT group to (8 ± 1.2) days (n = 18) (P < 0.001); simvastatin combined with aspirin delayed pathological changes of the transplanted hearts and protected vascular damage; simvastatin combined with aspirin upregulated eNOS and enhanced NO secretion. The level of CD4+CD25+Tregs in the blood of HT + A + S rats was significantly increased (2.2 ± 0.5)%, (2.9 ± 0.8)%, (4.3 ± 1.0)%, (8.3 ± 1.7)% and (14.3 ± 3.7)% for sham, HT, HT + S, HT + A and HT + A + S group respectively, HT vs. HT + A (P < 0.05) or HT + A + S (P < 0.01). Conclusion Simvastatin combined with aspirin delays the development pathology of myocardial and vascular damage and prolongs the survival time of cardiac allograft. The responsible mechanism is associated with activating CD4+ CD25+ Treg cells induction immune tolerance and enhancing vascular endothelial cell protection.
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Objective To investigate the expressions of Th17, CD4 +CD25 +Treg, HLA-DR mRNA in peripheral blood of children with EV71-induced hand, foot and mouth diseases ( HFMD ) and their clinical significance.Methods Stratified random sampling was used to select 60 children with HFMDs from Liaocheng People’s Hospital from February to October, 2014, including 20 mild, 20 severe and 20 critically ill cases.Twenty healthy children were also enrolled as the control group.All the children with HFMDs were given ribavirin (10 mg/kg) for the treatment.The percentages of Th17 and CD4 +CD25 +Treg cells in CD4 +T cells of peripheral blood were determined by flow cytometry, and the expression of HLA-DR mRNA in peripheral blood mononuclear cells was detected by reverse transcription polymerase chain reaction ( RT-PCR).Analysis of variance and SNK-q test were used to compare the expressions of Th17, CD4 +CD25 +Treg and HLA-DR mRNA among groups, and Pearson correlation analysis was performed to reveal the correlations between HLA-DR mRNA and Th17, CD4 +CD25 +Treg.Results Compared with the control group, the expression of Th17 was increased, while CD4 +CD25+Treg and HLA-DR mRNA expressions were decreased in children with HFMDs on d1 of treatment (F=310.4, 81.5 and 545.4, P0.05), but Th17 level in fatal was still higher and CD4 +CD25 +Treg level was still lower than those in control group (t=16.4 and 12.0, P<0.05).After 10 d of treatment, HLA-DR mRNA expressions in mild group and severe group were increased to the normal level.HLA-DR mRNA expression in surviving patients of critical ill group was still lower than that in mild group and severe group (P<0.05), but was higher than that in fatal patients (t=7.8, P<0.05).Pearson correlation analysis showed that, HLA-DR mRNA was negatively correlated with Th17 level (r=-0.770, P<0.01), and positively correlated with CD4 +CD25 +Treg level (r=0.883, P<0.01).Conclusion The expressions of Th17, CD4 +CD25 +Treg cells, and HLA-DR mRNA are correlated with the severity of HFMD, and may be used for evaluation of disease severity and prediction of disease outcomes.
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Objective To investigate the influence of coix seed triglyceride combined with transcatheter arterial chemoembolization (TACE) therapy on AFP, CD4﹢CD25﹢regulatory T (Treg) cells and cellular immune function in patients with advanced primary hepatocellular carcinoma (HCC). Methods A total of 50 patients with inoperable HCC, whose imaging examination showed no distant metastasis, were divided into the study group (n=25) and the control group (n=25). Coix seed triglyceride together with TACE was employed for the patients of the study group, while only TACE was adopted for the patients of the control group. For the patients of the study group, transcatheter hepatic artery infusion of 100 ml coix seed triglyceride was carried out during the performance of TACE, and postoperative intravenous drip of coix seed triglyceride (200 ml/d) was used for 5 days. The peripheral blood samples were collected one week before and one month after the treatment to detect the changes of AFP and T lymphocyte subsets (CD3﹢, CD4﹢, CD8﹢,CD4﹢/CD8﹢ and Treg) levels. One month after the treatment, enhanced CT, MRI or PET-CT was performed to evaluate the necrosis degree of the tumor. Results After the treatment, AFP levels was decreased in both groups, when compared the preoperative data the differences were statistically significant (P0.05). In the study group, the percentage of Treg cells decreased from preoperative (8.27±6.65)%to postoperative (4.22± 1.59)%, the difference was statistically significant (P0.05). Conclusion In treating advanced primary HCC, coix seed triglyceride combined with TACE can reduce the percentage of Treg cells, thus, influence the patient’s cellular immune status and possibly decrease the recurrence rate of HCC after TACE therapy.
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The airway remodeling of bronchial asthma is the result of repeated airway inlfammation. Its occurrence is a complex process involving many cytokines, inlfammatory mediators and associated cellular components, of which leukotrienes are important mediators of inlfammation in the airway remodeling. Many factors inlfuence Airway remodeling. In recent years, effects of Th17 cells and CD4+CD25+regulatory T cells (CD4+CD25+treg cells) on airway remodeling is growing in importance. Leukotriene receptor antagonist is an effective drug in the treatment of asthma and can suppress airway remodeling. But the exact mechanisms and its impact on the proportion of Th17 cells/CD4+CD25+treg cells is not yet clear. Therefore, the clariifcation of the changes of Th17 cells/CD4+CD25+treg cells expression in airway remodeling and the speciifc pathways, biological effects, inlfuence of the proportion of Th17 cells/CD4+CD25+treg cells expression after leukotriene receptor antagonist intervene can provide a new target for prevention and the treatment of asthma in the future.
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Immune thrombocytopenia (ITP), the most common hemorrhagic disease, is an organ-specific autoimmune disease characterized by decreased platelets count due to auto-antibodies mediating platelets destruction and insufficient platelets production. The etiology of ITP is still not completely known. Regulatory T cells, also called Tregs, are character-ized by CD4+CD25+, and positive of transcription factor forkhead box P3. They belong to a subpopulation of T cells special-ized for immune regulation and play an important role in maintaining the immune balance. Decreased production and defect-ed function of CD4+CD25+Treg was found not only in the ITP animal model but also in the ITP patients. It indicates that the Treg was involved in the pathology of ITP. This review focus on the characteristic and function of Tregs and their relationship with pathogenesis of ITP.
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This study was aimed to explore the effect on immunological function of T lymphocyte subpopulation and erythrocyte by the method of promoting blood flow and dissipating phlegm of rat with chronic pelvic inflammatory dis-ease (PID). A mixture of bacteria combined with mechanical injury was used in the establishment of a total of 75 Wistar chronic PID rat models. The rats were divided into the normal group, sham-operation group, model group, Gui-Zhi Fu-Ling Capsule (GZFLC) group, Shao-Fu Zhu-Y u Capsule (SFZYC) group, Jin-Gang-Teng Capsule (JGTC) group, and the control group of GZFLC, SFZYC, and JGTC. Intragastric administration of each medication was given according to different groups. The percentage of amount of CD3+, CD4+, CD8+and CD4+CD25+regulatory T cell (Treg) in the spleen of rats was observed in each group. The RBC-C3bRR and RBC-ICR in the blood serum of rats were also observed. The results showed that medications used in all treatment group significantly increased the percentage of amount of CD3+, CD4+ and CD4/CD8 ratio (P< 0.05) and reduced the percentage of amount of CD8+(P< 0.05). GZFLC has significantly decreased the abnormal increased percentage of the amount of CD4+CD25+ Treg. GZFLC and SFZYC significantly increased RBC-C3bRR (P< 0.05) and decreased RBC-ICR (P< 0.05). It was con-cluded that the prescription of promoting blood flow and dissipating phlegm can treatment chronic PID by adjusting immunity of T lymphocyte subpopulation and erythrocyte.
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Objective:To investigate the quantification of CD4~+CD25~+ regulatory T cells and distribution of CD4~+CD8~+ T lymphocyte subgroup in peripheral blood of patients in chronic hepatitis B (CHB),and to reveal relationship between CD4~+CD25~+ regulatory T cells,CD4~+CD8~+ T lymphocyte subgroup and HBV infetion as well.Methods:CD4~+CD25~(high),CD4~+CD25~+Foxp3~+Treg and CD3~+CD4~+CD8~+T lymphocyte subgroup in peripheral blood from 50 patients with CHB and 20 healthy controls was analyzed using flow cytometry.HBV DNA was detected by fluorescence quantitative PCR.Results:The number of CD4~+CD25~(high)Tregs in patients with CHB was obviously higher than that in healthy controls(P＜0.01)and increased with copies of HBV DNA.The same with the change of CD4~+CD25~+Foxp3~+Tregs in patients with CHB and there was a positive correlation between CD4~+CD25~(high)Tregs and CD4~+CD25~+Foxp3~+Tregs(r=0.890,P＜0.001).Compared with healthy controls,the frequency of CD4~+T cells and the ratio of CD4~+/CD8~+ in patients with CHB was declined,but there was no significant difference in the frequency of CD3~+T cells and CD8~+T cells between them(P＞0.05).The variation in the number of CD4~+CD25~(high)Tregs was correlated positively with the copies of HBV DNA(r=0.782,P＜0.001)and glutamic-pyruvic transaminase(ALT)(r=0.432,P＜0.005)separately,but negatively with the frequency of CD3~+,CD4~+,CD8~+T cells and the ratio of CD4~+/CD8~+(P＞0.05).The variation in the frequency of CD3~+,CD4~+,CD8~+T cells and the ratio of CD4~+/CD8~+ was also correlated negatively with the copies of HBV DNA(P＞0.05).Conclusion:The number of CD4~+CD25~(high)Tregs increases in patients with CHB and is in accordance with the copies of HBV DNA and increased level of ALT.Further studies should be done to investigate weather CD4~+CD8~+ T lymphocyte subgroup could be used to monitor the state of community.
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Objective:To explore the influence of tumor cells on CD4~+ CD25~+ Treg cells via TLRs.Methods:The numbers changes of CD4~+ CD25~+ Treg cells in the co-culture system of Lewis lung cancer cells and splenic lymphocytes were detected by flow cytometry;The expression of Foxp3 and TLR1-9 mRNA after co-culture were detected by RT-PCR;TLR9 expression on Lewis lung cancer cells was blocked by TLR9 receptor antagonist chloroquine.Results:Compared with control group,the number of CD4~+ CD25~+ Treg cells and Foxp3 mRNA expression were significantly increased in the co-culture group (P＜0.05).The expression of a variety of TLRs were affected by the co-culture of lymphocytes with Lewis lung cancer cells,and TLR9 mRNA expression was significantly increased compared with that of the control group (P＜0.05);Blocking TLR9 of Lewis lung cancer cells significantly reduce CD4~+ CD25~+ Treg cells and Foxp3 mRNA (P＜0.05).Conclusion:Lewis lung cancer cells could increase both number and function of CD4~+ CD25~+ Treg cells through inducing TLR9 expression in immunocells.This might be one of mechanisms of tumor-induced immune tolerance,and by which to contribute to the occurrence and progression of tumors.
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Objective To investigate the influence of pinealectomy(Px)and melatonin (MLT) supplementation on CD4~+CD25~+Treg cells development and Foxp3 expression in rat thymus. Methods One hundred and twenty clean-grade male SD rats were divided into five groups: normal control group, sham pinealectomized group, pinealectomized group, Px +7.5 mg/(kg·d) MLT group, Px +15 mg/(kg·d) MLT group, and the thymus were taken out at the 4th week and the 8th week;Flow cytometric analysis was used to analyse the number of double positive cells in the thymus and peripheral blood;Semiquantitative RT-PCR and immunohistochemistry were applied to analyse Foxp3 expression of rat thymus. Results There was no difference of the number of CD4~+CD25~+ Treg cells in rat thymus between Px and normal/sham group, and the number increased dependently on time and dose after MLT supplementation. In rat peripheral blood the double positive cells significantly increased in models of Px at the 4th week, and then backed to normal level after MLT supplementation. The results showed no significant changes in all groups at the 8th weeek;At the 4th weeks, Foxp3 expression increased in the thymus of Px rats compared to nomal/sham group, which returned to normal level after MLT supplementation. On the other hand, Foxp3 expression showed no significant difference in all groups at the 8th weeek.Conclusion Px made no difference between the development of CD4~+CD25~+Treg cells in rat thymus, but resulted in significant increase of thymic output and Foxp3 expression. All the effects disappeared at the 8th week. MLT supplementation could reverse the abnormity.
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Introducción: El Herpes Labial Recurrente supone una condición inmunológica alterada, tal como una hiperactividad de células T-reguladoras CD4+CD25+ (Treg). Éstas ejercen control sobre la tolerancia periférica y reducen el riesgo inmunopatológico, suprimiendo otras líneas celulares. Por ende, la supresión ejercida sobre la reacción inmune antiviral podría afectar negativamente el curso de la infección. Este contexto ha impulsado la búsqueda de nuevas alternativas inmunomoduladoras como la Equinácea purpúrea. Dada su propiedad inmunosupresora, se propone en el tratamiento del Herpes Labial Recurrente. Metodología: Estudio clínico prospectivo que analiza las subpoblaciones linfocitarias en 12 pacientes con Herpes Labial Recurrente, antes y después de recibir Equinácea purpúrea (30 gotas tres veces al día durante siete días).Resultados: En comparación con individuos sanos, los pacientes presentan una respuesta aumentada de células Treg. Esta condición se reduce significativamente tras recibir Equinácea purpúrea (515 + 145 y 432 + 113 cel/mm3 antes y después del tratamiento, respectivamente, p < 0,005). Conclusión: La hiperactividad de células Treg podría explicar el estado de inmunosupresión de estos pacientes y favorecería la persistencia viral. Se propone esta fitomedicina como una alternativa inmunoterapéutica beneficiosa.
Background: Recurrent Herpes Labialis patients may suffer from immunological alterations, such as CD4+CD25+Regulatory-T Cell (Treg) hyperactivity. These cells control peripheral tolerance and reduce immunopathology risk by suppressing other immunological cells. Hence, the Treg cell suppression on the antiviral immune reaction may perturb adversely the herpes infection outcome. This scenario has forced physicians to explore new immunomodulatory alternatives in Phytomedicine, such as Echinacea purpurea. Regarding the immunosuppressive property, it has been challenged to be employed in the Recurrent Herpes Labialis management. Methods: Clinical prospective study that analyzed lymphocytic subpopulation profile in twelve patients with Recurrent Herpes Labialis, before and after receiving E. purpurea (30 drops three times a day during seven days). Results: Comparing to healthy subjects, patients presented an enlarged Treg cell response. This condition became significantly reduced after receiving E. purpurea. (515 + 145 and 432 + 113 cel, before and after treatment respectively, p < 0.005). Conclusion: The intensified Treg cell activity may elucidate the immune suppression these patients undergo, aiding the viral persistence and survival. This proposes E. purpurea asa beneficial immunotherapeutic alternative.
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Humans , Antiviral Agents/therapeutic use , Echinacea/therapeutic use , Echinacea/chemistry , Herpes Labialis/drug therapy , Plant Preparations/therapeutic use , Antiviral Agents/pharmacology , Echinacea/pharmacology , Herpes Labialis/immunology , Herpes Simplex/drug therapy , Herpesvirus 1, Human , Immunomodulation , Multicenter Studies as Topic , Prospective Studies , Plant Preparations/pharmacology , Recurrence/prevention & control , T-Lymphocytes, RegulatoryABSTRACT
Objective To detect the ratio of CD4+ and CD8+ T cells and the number of CD4+CD25+ T regulatory cells(Treg)in human peripheral blood mononuclear cells(PBMC)of rheumatoid arthritis(RA)patients and evaluate the significance for diagnosis of RA.Methods The number of CD4+,CD8+ T cells and Treg cells were detected by flow cytometry and the ratio of CD4/CD8 was calculated.Results The ratio of CD4/CD8 and the number of CD4+CD25+ T cells detected in 49 RA patients were significantly higher than that in health group with statistical significance(P
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Objective:To investiate biological properties of Mesenchymal stem cell(BMSC) from rat bone marrow in vitro,and their effects in inducing immune tolerance.Methods:BMSCs were obtained from rat bone marrow by density gradient centrifugation and selected by cell attachment.The morphology of BMSCs was observed by electron microscope.The expression of surface molecules of CD34 and CD44 was analyzed by flow cytometry.The cells were injected ex vivo intravenously after in vitro culture and then CD4+ CD25+ Treg ratio were determined.The proliferation of thymic cells was estimated by 3H-TdR incorporation method.Results:BMSCs were fibroblast-like cells.The expression ratio of CD44 in BMSCs was 98%,but expression of CD34 was negative.After administration of BMSCs,the ratio of CD4+CD25+Treg(2.5%?0.69%) from peripheral blood of rats was higher than in normal group(0.8%?0.14%) and PBS control group(1.0%?0.23%),P
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Objective:To investigate the Foxp3 expression in murine model of type 1 diabetes mellitus and the effects of Foxp3 in the pathogenic mechanism of type 1 diabetes mellitus.Methods:Type 1 diabetes mellitus of mouse was induced by STZ.The Foxp3 expression in the spleen cells was detected at the mRNA level by RT-PCR and at protein level by Western blot.The percentage of CD4+CD25+ Treg cells in the spleens were detected by Flow cytometry.Results:The expressing levels of Foxp3 mRNA and scurfin in the model group was higher than those of control group within the first week after induction,but the expressing level of Foxp3 mRNA and Scurfin began to decrease on day 7 and were lower than those of control group on day 30.The percentage of CD4+CD25+ Treg cells in model group was similar with that of control group within the first week after induction,but after day 7,the percentage of CD4+CD25+ Treg cells in model group began to get lower than contol group.Conclusion:The expressing level of Foxp3 is decreased,then the proportion of CD4+CD25+ Treg cells is decreased accordingly,which may contribute to the pathogenic mechanism in type 1 diabetes mellitus.
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Objective:To explore the influence of tumor cells on CD4+CD25+Treg cells via TLRs.Methods:The numbers changes of CD4+CD25+Treg cells in the co-culture system of Lewis lung cancer cells and splenic lymphocytes were detected by flow cytometry;The expression of Foxp3 and TLR1-9 mRNA after co-culture were detected by RT-PCR;TLR9 expression on Lewis lung cancer cells was blocked by TLR9 receptor antagonist chloroquine.Results:Compared with control group,the number of CD4+CD25+Treg cells and Foxp3 mRNA expression were significantly increased in the co-culture group(P