Abstract:Foxp3+ regulatory T cells （Foxp3+Treg） are a special subset of T cells that prevent other immune cells from attacking the body’s own tissues, and are critical for maintaining immune homeostasis. The forkhead family transcription factor Foxp3 is the master regulator of Foxp3+Treg development and differentiation as well as its functional stability. The alteration of functional stability of Treg cells caused by the changes of Foxp3 protein level has been actively involved in controlling major human diseases including infectious diseases， autoimmune diseases， anaphylactic disease， tumor progression， tumor metastasis， and transplantation immunity. Understanding the function of Foxp3 in regulatory T cells differentiation and development and its functional stability will lead to novel therapeutic approaches for relevant immunological diseases.

Abstract:CD4+CD25+FoxP3+ regulatory T cell （Treg） plays an indispensable role in the maintenance of immune balance. In current clinical trials， Treg infusion has been proved to be an effective way to treat graft-versus-host disease （GVHD）. Therefore， how to improve Treg function， proliferation and survival by appropriate means is very important for basic and clinical immunology researchers. MicroRNAs （miRNAs） are a class of non-coding RNAs that can negatively regulate mRNA and inhibit the expression of target proteins. Several miRNAs have been shown to influence Treg function directly or indirectly. Here， we provide an overview of the relationships between these discovered miRNA signaling pathways and Treg cells， which might give a new insight into future basic and clinical studies.

Abstract:Regulatory T cell（Treg） is a thymus-derived T lymphocyte lineage with the ability of immunosuppression in the periphery， and it plays a key role in autoimmune diseases， immune tolerance， immune system， tumor immunity， transplantation immunity， allergic reaction and many other pathological immune processes. Natural Treg cell （nTreg） or called thymus regulatory T cell （tTreg） is a very important T cell lineage in Treg cells. In this paper，we make a review of this T cell lineage on the process of their differentiation，development and proliferation and the regulatory mechanism of them.

Abstract:Objective：To explore the influence of histone deacetylase inhibitor （HDACi） trichostain A （TSA） on the expression of interferon regulatory factor 3 （IRF3） and the preliminary epigenetic mechanism of transcriptional regulation. Methods：Luciferase assays were applied to detect IRF3 promoter activity after TSA incubation and histone acetyltransferases （HAT） p300 transfection. The IRF3 mRNA expression level was detected by Real-time fluorescence quantification-PCR， while the IRF3 protein expression level was detected by Western blot. Results: IRF3 gene promoter activity was increased after TSA intervention at different concentrations. TSA treatment （1 μmol/L） induced the relative luciferase activity （RLA） of IRF3 promoter PS1 and PS6 by 75% and 155%， respectively. Post-transfection with p300 increased the RLA of PS1 by 267%. At the 1 μmol/L and the 10 μmol/L TSA group， the IRF3 mRNA expression was increased by 23% and 39% after 6 h treatment， while increased by 7% and 64% after 24 h， respectively. The IRF3 protein expressions were increased by 15%， 72%， and 191% after TSA （0.1 μmol/L，1 μmol/L， and 5 μmol/L） treatment， respectively. Conclusion：TSA can up-regulate the expression of IRF3 both on mRNA and protein levels， while TSA and p300 can increase transcription activity by targeting IRF3 promoter region， which suggests that HDAC and acetylation are positive regulation of IRF3 transcription or expression， and indicates the hypothesized role of TSA in the immunotherapy and antitumor effects through IRF3 pathway.

Abstract:Objective：To investigate the effect of X-box binding protein 1 (XBP1) on embryogenesis of Xenopus laevis. Methods： The expression of XBP1 during each stage of embryogenesis was detected by Western blotting assay， the whole mount in situ hybridization，and immunostaining. The micro-injection of morpholino (MO) was performed to knockdown XBP1. The expression of marker genes was tested during germ layer formation，as well as embryo development by whole mount in situ hybridization and real-time PCR. Results： XBP1 expressions of the nerve，anterior kidney，pancreas，and other organs were detected during embryogenesis of Xenopus laevis. Knockdown of XBP1 inhibited the expression of germ layer related genes at the early stage of embryogenesis，and then inhibited pancreas development related gene in Xenopus laevis embryos at the late stage of embryogenesis. Conclusion ：XBP1 may be required for the germ layer formation at the early stage of embryogenesis and pancreas development at the late stage of embryogenesis.

Abstract:Objective: To observe the effects of endotoxin tolerance induced by lipopolysaccharide （LPS） on the production of anti-inflammatory cytokine IL-10 and the level of apoptosis in human neutrophils. Methods: Neutrophils were pretreated with 1 μg/mL Porphyromonasgingivalis （P.gingivalis） LPS or 1 μg/mL Escherichia coli （E.coli） LPS for 12 h to induce cell tolerance. Then， the cells were washed and stimulated with the same LPS for additional 20 h （for ELISA） or 3 h （for flow cytometry）. Levels of IL-10 in supernatants and apoptosis of neutrophils were detected by ELISA and flow cytometry， respectively. Results: After 20 h， the amount of IL-10 secreted by neutrophils treated with P.gingivalis LPS or E.coli LPS was increased significantly compared with that without any stimulation （P<0.05）， while no significant changes of apoptosis in neutrophils with LPS stimulation were observed after 3 h （P>0.05）. After restimulation with P.gingivalis LPS or E.coli LPS， IL-10 production was increased significantly compared with that secreted by neutrophils challenged with the same LPS only once （P<0.05）. However， no significant changes of apoptosis in the short term were detected after P.gingivalis LPS retreatment for 3 h （P>0.05）， and apoptosis levels in the short term were significantly decreased after E.coli LPS restimulations for 3 h （P<0.05）. Conclusion: Endotoxin tolerance might contribute to the increased production of IL-10 in human neutrophils， but the effects of tolerance on apoptosis might depend on the types of LPS.

Abstract:Objective: To study the mechanism of toxic effect of polychlorinated biphenyls PCB1254 on retinal ganglion cells （RGC-5）. Methods: After RGC-5 cells were exposed to 0.125， 0.250， 0.500， 1.000 mg/L PCB1254， 0.01% methanol， and pure water， the level of miR-182 was detected in every group. In addition， by using the miR-182 mimics or miR-182 inhibitor to up-regulate or down-regulate the expression of miR-182 in RGC-5， apoptosis， proliferation，and cell cycle were observed. After the treatment with 0.5 mg/L and 1.0 mg/L PCB1254， miR-182 mimics were transfected into RGC-5 cells to observe the apoptosis and proliferation. Results: ① With the increasing concentration of PCB1254， the expression of miR-182 was declined. When the concentration was ≥0.5 mg/L， there were significant differences between experimental groups and the control group （P<0.05）. ② MiR-182 silencing led to increased Caspase-3 activity in RGC-5 cells， which indicated that miR-182 silencing promoted apoptosis （P<0.05）. CCK-8 assay showed that the cell viability was lower in miR-182 inhibitor group than that in the negative control group， which indicated that miR-182 silencing inhibited cell proliferation（P<0.05）. There were no significant differences in cell cycle between the negative control group and the miR-182 inhibitor group（P>0.05）. ③ When miR-182 were tranfected in RGC-5 exposed to 0.5 and 1.0 mg/L PCB1254， the caspase-3 activity decreased（P<0.05）, and cell viability increased （P<0.05）. It showed that miR-182 could improve the toxic effect of PCB1254 in cell apoptosis and proliferation. Conclusion: Our findings suggest that the PCB1254 may affect the proliferation and apoptosis of RGC-5 by inhibiting the expression of miR-182， which may lead to the damage of visual function.

Abstract:Objective: To investigate the effects of CYP2C9 and CYP2A6 genetic polymorphisms on plasma concentrations of sodium valproate （VPA） in the epileptic children． Methods: Epileptic children treated with sodium valproate only were collected in our study. Fluorescence polarization immunoassay was performed to measure plasma concentrations of sodium valproate. Direct sequencing and nest-PCR were applied to identify the genotypes of CYP2C9 and CYP2A6. One-way ANOVA was performed to analyze the influence of the polymorphisms on plasma concentrations of sodium valproate. Results: Patients were divided into 3 groups according to the genotypes of CYP2C9 and CYP2A6: extensive metabolizers （EM， CYP2C9*1*1 & CYP2A6*1*1）， intermediate metabolizers （IM， CYP2C9*1*3 & CYP2A6*1*1 or CYP2C9*1*1 & CYP2A6*1*4）， and poor metabolizers （PM， CYP2C9*1*3 & CYP2A6*1*4 or CYP2A6*4*4）， and the frequencies of the three groups were 73.5%， 24.1%， and 2.4%， respectively. The standardized blood drug concentration of IM was significantly higher than that of EM （P<0.05）. The mean of standardized blood drug concentration of PM was higher than the others， but there were no significant differences between them （P>0.05）. Conclusion: The plasma concentrations of sodium valproate can be affected by the polymorphisms of CYP2C9 and CYP2A6. Clinicians can choose appropriate initial dosage by detecting the genotypes.

Abstract:Objective: To investigate the inhibitory effect and mechanism of recombinant analgesic-antitumor peptide （rAGAP） enhanced 5-fluorouracil（5-FU） on H22 hepatoma. Methods: The models of H22 hepatoma in mice were established through ascites inoculating H22 cells suspension into mice in right armpits， and randomly divided into 4 groups. rAGAP group was given 0.03 mg/kg IP rAGAP， 5-FU group 10 mg/kg IP 5-FU， United group 0.03 mg/kg 5-FU and 10 mg/kg IP rAGAP， Model group used equal volume of normal saline intraperitoneal injection for 3 weeks. After that， the tumor tissue were sampled， the tumor weight and tumor inhibition rate were detected. The expressions of PI3K， AKT， and PTEN were detected by Western blot method. Results: Compared with Model group， mice tumor weight of other groups was significantly lower （P<0.05）; tumor suppressor rate of United Group was obviously higher than that of rAGAP group and 5-FU group （P<0.05）. Compared with Model group， PI3K and p-Akt expression of other groups were significantly decreased， but PTEN expression was significantly increased （P<0.05）; compared with 5-FU group， rAGAP group， PI3K and p-Akt expression of United group were significantly reduced， but PTEN expression was significantly increased （P<0.05）. Conclusion: rAGAP can enhance the inhibitory effect of 5-FU on H22 hepatoma， its mechanism may be related to the activity of PI3K/AKT/PTEN signaling pathway， and then inhibition the proliferation of H22 hepatoma.

Abstract:Objective：To study the function of angiogenin and other inflammatory mediators in rats with acute biliary pancreatitis （ABP）. Methods：Seventy SD rats were randomly divided into the ABP group （n=56， ligating bile pancreatic duct） and the SO （sham operation， only flipped pancreas） group （n=14）. The rats were sacrificed at 0， 6， 12， 24， 48， 72， and 120 hours after operation. The samples of blood serum and pancreas tissues were collected at each time point for detecting AMY， IL-10， TNF-α， NF-κB， Ang-1， Ang-2 levels using ELISA， pathological score and pancreatic moisture content. Results：The levels of AMY， IL-10， TNF-α，NF-κB at each time point in the ABP group were significantly higher than those in the SO group （P<0.05）， and reached their peak value at 120 h during the observation period. The level of Ang-1 in the ABP group at 0 h decreased significantly（P<0.01）， getting a little higher and remaining stable after 12 h. Ang-1 had no relationship with the severity of ABP. Ang-2 in the ABP group increased from 0 h， reaching its peak value at 48 h， and decreased at 72 h and 120 h. The level of Ang-2 in the ABP group was significantly higher than that in the SO group at each time point （P<0.01）. Moreover， the level of Ang-2 was significantly positively correlated with ABP pathological score at the first 48 h （r=0.943， P<0.01）， but the relationship was lost after 48 h. Similarly， the level of Ang-2 was also correlated with pancreatic moisture content during 12 h （r=0.830， P<0.01）. Moreover， at first 6 h， pancreatic moisture content was significantly increased， which was corresponding to the decrease of Ang-1 and the increase of Ang-2. Conclusion：Inflammatory mediators play an important role in ABP. Ang-1 had no relationship with the severity of ABP. Elevated Ang-2 was significantly positively correlated with ABP pathological score and pancreatic moisture content in the early stage of ABP. Ang-2 may play a significant role in promoting vascular leak and proinflammatory effect in early stage of ABP， which indicates the severity of pancreatitis， or can be used to guide clinical treatment.

Abstract:Objective: To determine whether p27 plays an important role in modulating bone mass and bone turnover. Methods: We used 8-weeks-old p27 homozygote （p27-/-） mice and WT littermate mice. Firstly， we performed bone marrow ablation （BMX） on both tibia and femur， and then we compared the difference between osteogenesis and bone turnover of the different groups by radiography and pathologic histology. Finally， we detected the difference of ostosis and bone turnover between groups on the 3rd/7th/11th/14th day after BMX respectively. Results: Three days after BMX， the bone mass of both WT and p27-/- littermate showed no new osteogenesis. While compared to WT littermate， on the 7th/11th/14th day after BMX， the new osteogenesis in p27-/- mice increased significantly. Further study showed that the number of osteoblasts and the positive area of type I collagen increased significantly. However， the number of osteoclasts and the positive area of osteoclasts decreased markedly. Conclusion: The BMX of p27 gene knockout mice study proved that p27 could reduce the bone mass and inhibit bone turnover by suppressing the osteogenesis of osteoblasts and promoting the bone resorption of osteoclasts.

Abstract:Objective: To investigate the effects of construction of the central vascularized artificial bone with β-tricalcium phosphote and explore the mechanism of vascularization in artificial bone for its further clinical application. Methods: We built a model by implanting the selected New Zealand rabbits lumbar dorsal artery side channel of the β-tricalcium phosphate， filled with the autologous tiny bone particles， and flushed the latissimus dorsi muscle bag. Pure artificial bone was put into every rabbit’s lift latissimus dorsi muscle bag as the control group. The samples were taken out for spiral CT， X-ray， and histology after 4， 8， and 12 weeks. Results: The site where the implanted vascular bundles appeared like foramen nutriens， the blood vessels were abundant， which emerged from deep tissue of the artificial bone were evident on the surface specimen， the central vascularized tricalcium phosphte were filled with new-born vessels at 4-8 weeks. Newly formed bone appeared after 4 weeks and was more mature at 12 weeks. There were only a few new-born vessels in control. Four weeks postoperatively， there were no significant differences on CT， X-ray， and histological grading between the experimental group and the control group （P>0.05）； 8， 12 weeks postoperatively， the difference was significant on CT， X-ray， and histological grading between the experimental group and the control group （P<0.05）. Conclusion: Putting an artery into central axis of the artificial bone and implanting the artificial bone into rabbit’s latissimus dorsi muscle can notably improve vascularization of artificial bone. This method can be used to build model of vascularized bone.

Abstract:Objective: To investigate CTLA-4 expression in T cells from peripheral blood of patients with systemic lupus erythematosus （SLE） and its significance in clinic. Methods: Peripheral blood mononuclear cells （PBMCs） from SLE patients and healthy donors were isolated and then cultured with anti-CD3 and anti-CD28 antibodies for 48 hours. Before and after culture， the cells were detected for the percentages of CTLA-4+ cells in CD4+ and CD8+ T subsets by flow cytometry （FCM）. Based on these data， the correlation between CTLA-4 expression in CD4+ or CD8+ T subsets of SLE patients and SLE disease activity index （SLEDAI） and kidney damage were analyzed. Cell culture supernatants were also collected for the determination of free CTLA-4 level by ELISA. Results: In fresh PBMC， CTLA-4+ cell percentages of CD4+CD25+ and CD8+CD25+ T subsets in SLE patients， were significantly higher than those in controls， and were positively correlated with SLEDAI; CTLA-4+ cell percentage of CD8+CD28- T subset was also significantly higher in SLE than that in controls， but was not correlated with SLEDAI. After stimulation with anti-CD3 and anti-CD28， CTLA-4+ percentages of CD4+CD25+， CD8+CD25+ and CD8+CD28- T subsets were significantly lower in SLE than those in controls， but was not correlated with SLEDAI. While in active SLE， CTLA-4+ cell percentage of CD8+CD28- T subsets was significantly lower in patients with kidney damage than that in patients without kidney damage; CTLA-4 level of the culture supernatant was lower in SLE patients than that in controls. Conclusion: The abnormally increased expression of CTLA-4 in T （CD4+ and CD8+） cells fresh isolated from SLE patients T cells might indicate disease development with T cell activation. On the other hand， T cells of SLE patients are deficient in induced expression of CTLA-4， which might be related with the deficient ability of reactivation of SLE T cells in vitro.

Abstract:Objective: To investigate the correlation between CD4+CD25+ and CD4+CD25+Foxp3+ regulatory T cells (Treg) and transforming growth factor-β1 （TGF-β1） in peripheral blood of patients with epithelial ovarian cancer. Methods: The percentages of CD4+CD25+ and CD4+CD25+Foxp3+ Treg in peripheral blood from 24 patients with ovarian cancer were detected by flow cytometry. Plasma level of TGF-β1 in each patient was detected by enzyme-linked immunosorbent assay （ELISA）， and compared with that in 20 patients with benign ovarian tumor and 20 healthy volunteers. Results: The percentages of CD4+CD25+， CD4+CD25+Foxp3+ Treg cells and TGF-β1 in ovarian cancer patients were significantly higher than those in 20 patients with benign ovarian tumor and 20 healthy volunteers （both P<0.01）. Levels of CD4+CD25+， CD4+CD25+Foxp3+ Treg cells and TGF-β1 in ovarian cancer patients at stage Ⅲ~Ⅳ were significantly higher than those of patients at stage Ⅰ~Ⅱ（P<0.05）. Pearson correlation analysis showed that the percentages of CD4+CD25+ and CD4+CD25+Foxp3+ Treg cells were positively correlated with the levels of TGF-β1. Conclusion: The percentages of CD4+ Treg cells in peripheral blood from ovarian cancer were higher and positively correlated with TGF-β1 levels. Detection of CD4+ Treg cells and TGF-β1 has the clinical value on evaluation of development and prognosis of different stages of ovarian cancer. The increased levels of TGF-β1 may be involved in the immune negative adjustment mechanism of CD4+ Treg cells.

Abstract:Objective: To investigate the diagnostic value of combined use of serum CA125 and plasma D-dimer in ovarian cancer. Methods: Totally 109 patients with ovarian tumor were randomly selected. According to the result of pathology examination， patients were divided into two groups， benign ovarian tumor group （59 cases） and malignant ovarian tumor group （50 cases）. Thirty healthy subjects were also selected as control group during the same period. The level of serum CA125 and plasma D-dimer was measured. Results: Combined use of serum CA125 and plasma D-dimer can improve the diagnostic accuracy of ovarian malignant tumors. The level of plasma D-dimer in advanced malignant ovarian tumor patients was significantly higher than that of patients with early malignant ovarian tumor. The level of plasma D-dimer in serous ovarian cancer was also significantly higher than that of patients with not serous ovarian cancer. The diagnostic accuracy of combined use of serum CA125 and plasma D-dimer in malignant ovarian tumor is higher， especially in serous ovarian tumors. Conclusion: D-dimer combined with CA125 can be used as a more effective marker of malignant ovarian tumors， especially advanced ovarian cancer.

Abstract:Objective: To investigate the diagnostic value of combining determination of ESM-1 and CEA levels in differentiating tuberculous（TPE） from malignant pleural effusion（MPE） caused by lung cancer. Methods: Totally 65 NSCLC patients with MPE and 45 patients with TPE were enrolled into this study. The ESM-1 level in pleural effusion was tested by ELISA and CEA levels were tested by the chemiLuminescence method. All results were analyzed by the statistical method. Results: The levels of ESM-1 and CEA in MPE was significantly higher than those in TPE（P<0.001）. The sensitivity of ESM-1 testing for diagnosing MPE was 81.5%， the specificity was 84.60% and the accuracy was 82.73%. The sensitivity of CEA testing was 73.80%， the specificity was 95.60%， and the overall accuracy was 82.73%. The sensitivity of the combined testing was 83.10%， the specificity was 96.30%， and the overall accuracy was 82.73%. The sensitivity and the overall accuracy of combined testing were higher than those by ESM-1 and CEA testing single. Conclusion: MPE presents higher ESM-1 concentration than TPE does，and determination of ESM-1 is useful to differentiate MPE from TPE. The combined testing of ESM-1 with CEA can increase the sensitivity of diagnosis of MPE.

Abstract:Objective: To compare the protection effects between side branch balloon submerged embedding technique and side branch guide wire protection technique on the side branch in the process of main branch stent implantation during PCI for true coronary bifurcations. Methods: A total of 44 patients with true coronary bifurcations were randomly divided into two groups: group A and group B. Twenty cases in group A were treated with side branch balloon submerged embedding technique and the other 24 patients in group B were treated with side branch guide wire protection technique. Several parameters were compared， including the damage rate of the ostium of the side branch， the severe damage rate of the ostium of the side branch， the loss rate of the side branch， guide wire exchange time， the X-ray exposure amount， contrast agent dosage， the rising rates of postoperative TNT-h， and the rate of postoperative ischemic chest pain. Results: There were no statistic differences in the damage rate of the ostium of the side branch， the loss rate of the side branch， and the rate of postoperative ischemic chest pain （all P>0.05）. The severe damage rate of the ostium of the side branch， guide wire exchange time， the X-ray exposure amount， contrast agent dosage， and the rising rate of postoperative TNT-h had statistical differences （all P<0.05）.　The incidence of major cardiovascular events （MACE） after 8 to 12 months follow-up had no significant difference（P>0.05）. Conclusion: During PCI for true coronary bifurcations， the side branch balloon submerged embedding technique can reduce the severity of the ostium of the side branch damage， increase the successful rate of guide wire exchanging， shorten operation time， reduce X-ray exposure amount， contrast agent dosage and myocardial damage compared with the side branch guide wire protection technique after main branch stent implantation.

Abstract:Objective: To establish a method for determination assay of empagliflozin and related substance. Methods：HPLC method was adopted. The determination was carried out on Waters symmetry C18 column（250 mm×4.6 mm，5 μm）. The mobile phase A was 0.01%（V/V） trifluoroacetic acid solution，the mobile phase B was acetonitrile，linearity gradient elution was as follows：0~10 min，mobile B 32%；10~50min，mobile B 32%~95%， at a flow rate of 1.0 mL/min. The detective wavelength was set at 225 nm， and the column temperature was at 45℃. Results：The separation between empaliflozin and its related substances was not less than 1.5. The LOQ was less than 9 ng/mL，and linear range were suitbale for determination. The RSD of repeatability was less than 5.0%. The test solution and reference solution had a good stability in 8 h， the recovery of the impurities was 88.6%~106.1%， and RSD was less than 10%. Conclusion：The method is sensitive and accurate， and is effective for quality control in empagliglozin.