目的 通过观察卵巢早衰 POF 患者外周血CD4+CD25+调节性T细胞 Treg 及干扰素-γ（IFN-γ）、转化生长因子-β(TGF-β)的变化，探讨POF的免疫学发病机制。 方法 收集2011年12月－2012年9月就诊的POF患者17例，卵巢储备功能减退 DOR 患者11例，以及生殖中心健康育龄女性16例，流式细胞仪定量检测外周血Treg数量，Elisa方法检测血清IFN-γ、TGF-β的水平，并以FSH/LH评价卵巢储备功能，进行相关性分析。 结果 与对照组相比，POF组和DOR组IFN-γ水平增高 P＜0.01 、TGF-β水平降低 P＜0.01 ，POF患者及DOR患者Treg比例降低 P＜0.01 ，IFN-γ的增高与卵巢储备功能的下降呈显著正相关 r=0.70，P＜0.01 。 结论 Treg 和IFN-γ、TGF-β水平与卵巢早衰密切相关，IFN-γ对评估卵巢储备功能、预测卵巢早衰具有参考价值。
ObjectiveTo investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice. MethodsTwelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23Rlentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. ResultsCompared with the LV-Ctrlgroup, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group (t=－2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23Rgroup were higher than those of LV-Ctrl group, and the difference was statistically significant (t=－4.429, －6.578, －7.768, －10.183, －6.325, －7.304, －4.841, －6.872; P＜0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant (t=－4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23RmRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant (Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant (t=－6.313,－5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant (t=－5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant (t=－4.290, 3.700; P=0.002, 0.006). ConclusionIL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.
ObjectiveTo investigate the change of cellmediated immunity in gut mucosa after major hepatectomy and to study its relationship with the bacteria translocation.MethodsFortyeight Spraguedawley adult male rats were randomly allocated into two groups, the sham operation group and the operation group. Besides without the hepatectomy, the sham operation group has the same course with the operation group. Seventy percent hepatectomy rats are divided as postoperative 6 h group (n=6)，12 h group (n=6)，24 h group (n=6) and 72 h group (n=6). Sixhour, 12hour, 24hour and 72hour after operation specimens were taken from jejunoileum respectively. Immunohistochemical staining was performed on frozen sections and image pattern analysis was used. We also investigate the change of liver function. ResultsTwentyfour hours and 72 hours after 70% hepatectomy, there was a significant reduction in the number of CD3+，CD4+and CD8+ T lymphocytes in the mucosal lamina propria of the operation group compared with the sham operation group （Plt;0.05）. There was significant difference between these two groups in liver function change （Plt;0.05）.ConclusionThere is an altered pattern of intestinal mucosal T lymphocytes after major hepatectomy, then the local cellmediated immunity was depressed, which may be the cause of translocation of enteric bacteria.
Diabetic retinopathy (DR) isacommon cause of blindness, its occurrence and development are the synergic results of multiple factors. Current studies suggest that inflammation and inflammatory factor has an important role in the pathogenesis of DR. The occurrence and development of DR are closely related with interleukins, intercellular adhesion molecules, hasten factors, tumor necrosis factor, C-reactive protein etc. Mesenchymal stem cells (MSCs) are pluripotent cells derived from the mesoderm and have multiple differentiation potentials, and anti-inflammatory and immunosuppressive function. Recent studies shown that MSCs transplantation can protect damaged retina by inflammatory regulation, which becomeanew research direction for DR treatment.
Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
Objective To explore the impact of recombinant human growth hormone （rhGH） on T lymphocyte subsets in patients with rheumatic heart disease during the perioperative period of heart valve replacement. Methods A total of 65 patients with rheumatic valvular heart disease who received heart valve replacement in Department of Cardiothoracic Surgery of Xiangyang Central Hospital from June 1， 2011 to March 31， 2012 were enrolled in this double-blind randomized controlled clinical study. All the patients were divided into 2 groups by random number produced by SAS software：the trial group and the control group. There were 35 patients in the trial group including 19 males and 16 females with their average age of 50.57 years， and 30 patients in the control group including 16 males and 14 females with their average age of 49.87 years. Apart from routine cardiac glycosides， diuretics， glucose-insulin-potassium solution， and postoperative anti-infective therapy， patients in the trial group also received subcutaneously injection of rhGH 5 U （1 ml）daily from 1 day before surgery to 3 days after surgery， and patients in the control group received subcutaneously injection of normal saline 1 ml as placebo. Peripheral venous blood samples were taken in the morning 2 days before surgery and 1 st， 3 rd， 7 th day after surgery respectively. Percentages of CD3+， CD4+， CD8+ were examined timely by flow cytometry and CD4+ /CD8+ ratio was calculated. Results In the control group， percentages of CD3+， CD4+ and CD4+ /CD8+ ratio on the 1st， 3rd， 7th postoperative day were significantly lower than preoperative levels， and percentages of CD8+ on the 1st and 3rd postoperative day were significantly lower than preoperative level （P＜0.05）. In the trial group， percentages of CD3+， CD4+， and CD8+ on the 1st and 3rd postoperative day were significantly lower than preoperative levels（P＜0.05）， while percentages of CD3+， CD4+， and CD8+ on the 7th postoperative day were not statistically different from preoperative levels （P＞0.05）; CD4+ /CD8+ ratio on the 1st postoperative day was significantly lower than preoperative level （P＜0.05）， while CD4+ /CD8+ ratios on the 3rd and 7th postoperative day were not statistically different from preoperative level （P＞0.05）. There was no statistical difference in preoperative T lymphocyte subsets between the trial group and the control group （P＞0.05）. The percentages of CD4+ and CD4+/CD8+ ratio in the trial group were significantly higher than those of the control group on the 1st postoperative day （P＜0.05）， while the percentages of CD3+ and CD4+ and CD4+ /CD8+ratio in the trial group were significantly higher than those of the control group on the 3rd and 7th postoperative day（P＜0.05）. Conclusion Use of rhGH can significantly increase T lymphocyte subsets expression， enhance body cellular immunity， and improve postoperative recovery of patients with rheumatic valvular heart disease during the perioperative period of heart valve replacement.
Objective To explore the possible anti-inflammatory mechanism of peroxisome proliferators-activated receptor（PPAR） gamma-agonists by investigating the effects of Rosiglitazone on the expression of phosphorylation of signal transducer and activator of transcription 6（p-STAT6） and the secretion of interleukin（IL）-4 in T-lymphocytes from patients with acute asthma.Methods Peripheral blood T-lymphocytes from 10 healthy volunteers（group A） and 10 patients with acute asthma were isolated，purificated and cultured.T-lymphocytes from the asthma patients were divided into a control group（group B） and a Rosiglitazone treated group（group C）.Rosiglitazone was added with a single dose of 10-4 mol/L at 0 hour of cultrue.After cultured for 48 hours，the concentration of IL-4 in supernatant of each groups were detected by ELISA.The express of p-STAT6 in the T-lymphocytes were determined by Western blot and immunohistochemical techniques.Results The levels of IL-4 were increased markedly in group B than those in group A and group C[（170.34±9.05）pg/mL vs（76.82±7.06）pg/mL and（123.59±8.70）pg/mL，both Plt;0.01]，and which in group C was significantly lower than group A（Plt;0.01）.The levels of p-STAT6 in T lymphocytes were increased markedly in group B than in group A and C[Western blot：（6.28±0.19 vs 3.07±0.18 and 4.12±0.16；immunohistochemistry：（36.58%±7.41）% vs（11.39±4.02）% and（23.92±5.8）%，all Plt;0.01），and which in group C were significantly higher than that in group B（both Plt;0.01）.There was a positive correlation between the level of p-STAT6 and IL-4（Plt;0.01）.Conclusion The levels of p-STAT6 and IL-4 in T-lymphocytes of patients with acute asthma were suppressed by Rosiglitazone in vitro.
ObjectiveTo evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection. MethodsBMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO[green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor). IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPMI 1640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. ResultsThe IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P<0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P<0.05); there was no significant difference between experimental group 4 and the positive control group (P>0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P<0.05) except experimental group 4 (P>0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5±0.6) days in group A, (14.5±0.8) days in group B, and (9.0±0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P<0.05). ConclusionIDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.
Abstract: Objective To investigate the influence of cardiopul monary bypass(CPB) to the cellular immune function of T lymphocyte. Me th ods Among 500 patients operated from March 2006 to September 2006，30 patients with rheumatic heart disease were selected randomly as the CPB group, which would replace mitral valve; 30 patients with congenital patent ductus arte reriosus as the nonCPB group, which would ligate ductus arteriosus without CPB . The blood was sampled before operation, at the end of CPB or operation, and 24 hours after operation. After T lymphocyte was seperated, the quantum o f T lymphocyte, apoptosis of T lymphocyte, ability of T lymphocyte to kill tumou r cell were measured. Results The quantum of T lymphocyte i n CPB group at the end of CPB was decreased than that before operation （50.9% ±6.8% vs. 58.5%± 9.1%,Plt;0.05）; apoptosis of T lymphocyte at the end of CPB and 24 hou rs after operation were increased than that before operation （6.5%±2.2% vs. 0. 9%±1.1%, 5.6%±1.8% vs. 0.9%±1.1%;Plt;0.01）; ability to kill tumour cell b reakdown in CPB group at the end of CPB and 24 hours after operation was decrea sed than that before operation （30.4%±6.0% vs. 37.3%±8.6%, 29.0%±4.9% vs . 37 .3%±8.6%;Plt;0.05）. Ability to kill tumour cell breakdown in CPB group was lower than that in nonCPB group at the end of CPB （30.4%±6.0% vs. 33.6%±5. 3%, Plt;0.05）. Conclusion CPB can depress the cellular im mune function,which causes temporary immune depression to the body.
Objective To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyteassociated antigen 4 immuno globlin(CTLA4-Ig). Methods Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection ofL6(as a control), was named AL group. The second group,also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group,homologous BALB/C with injection of PBS buffer solution, was named AB group.The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6(BC group) and C3H(BHgroup) mice respectively after first 12 mice being transplantated with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. Results Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets(Plt;0.05), the serum antibody(Plt;0.05) and lymphocyteproliferation of the second stimulation by splenic cell and bone supernatant ofdonor (Plt;0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4T cell subsets(Pgt;0.05), the serum antibody (Pgt;0.05), and lymphocyte proliferation of the second stimulation(Pgt;0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared,cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (Plt;0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor(Plt;0.05), BC group was significantly lower than those of BH group. So theimmunological tolerance induced by CTLA4-Ig was of donor-specificity. Conclusion The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.