Background Researches showed that CD4+CD25+ natural regulatory T cells (nTregs) play an important role in maintaining peripheral immune tolerance, while immunotherapy using in vitro-expanded induced regulatory T cells (iTregs) suppresses allograft rejection in multiple organ transplantation.The inducing method of iTregs still needs to be optimized.Furthermore,the effect of iTregs on grafts of keratoplasty is unclear.Objective This study was to investigate the inducing and expansion method of iTregs and explore its inhibitory effects on corneal allograft rejection.Methods Bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice femora and cultured.CD4+ CD25+ T cells and CD4+ CD25-T cells were isolated from mouse spleen and separated using flow cytometry.The CD4+CD25-T cells were divided into negative control group (CD4+CD25-T cells), CD3/ 28 antibody bead group (CD4+CD25-T cells+CD3/28 antibody bead) ,2.5 ng/ml transforming growth factor (TGF)-β1 induced group and 10.0 ng/ml TGF-β1 induced group.The iTregs was formed after induction of different concentrations of TGF-β1 and CD3/CD28 antibody bead (1 : 1).CD3/CD28 antibody bead (1 : 2) , interleukin-2 (IL-2) and TGF-β1 were used to expand iTregs.The phenotype and proliferation of iTregs were assayed by flow cytometry,and the inhibitory effect of iTregs on effector T cells (Teffs) was analyzed by mixed lymphocyte reaction.Allogenic keratoplasty model (C57BL/6→BALB/c) was build,and 0.1 ml iTregs or nTregs suspension or PBS was injected via posterior venous plexus of fellow eyes to assess the graft survival time.The use and care of the mice followed the ARVO statement.Results The CD4+CD25+ T cell proportions were (6±3)％ ,(91±4)％ ,(91±3)％ and (86± 6) ％ in the negative control group,CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1induced group, showing significant increases in the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the negative control group (all at P＜0.01).The Foxp3+ T cell proportions of the CD3/CD28 antibody bead group,2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group were (1.18 ±0.20) ％ , (8.70± 1.80) ％ and (21.80±3.36) ％ , showing significant increases in the 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the CD3/CD28 antibody bead group (both at P＜0.01).Compared with the nTregs, the expression of CD69 was lower, and the expressions of PD-1 and Foxp3 were raised in the iTregs (all at P＜0.01).The proliferation of Teffs were decreased when cocultured with iTregs in comparison with nTregs at 1 : 1,1 : 2,1 : 4,1 : 8,1 : 16 Tregs/Teffs rations (all at P＜ 0.01).The survival time of mouse corneal grafts was 4 weeks with the permanent tolerance of 50％ in the iTregs injected group,which was superior to the 3 weeks survival time and 17％ permanent tolerance in the nTregs injected group(P＜0.05).Conclusions TGF-β1 can induce CD4+ CD25-T cells to form iTregs, which highly express Foxp3.iTregs show a stronger inhibitory effect on the growth of lymphocytes than nTregs, and therefore suppress the graft rejection after keratoplasty.

Objective To explore the correlation between expression of VEGF in colorectal cancer and pathological factors.Methods The expression of VEGF in 99 cases with colorectal cancer was examined by immunohistochemical staining.Results It showed that the expression of VEGF in colorectal cancer was correlated with depth of invasion and lymph node metastasis and was not correlated with tumor region,and histologic type.The frequency of positive cases in patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis(P

Objective To analyze the clinical and histopathological features,immunophenotypes,treatment and prognosis of subcutaneous panniculitis-like T cell lymphoma (SPTL).Methods Clinical and experimental data were collected from 9 cases of SPTL,and retrospectively analyzed.Related pathological and immunohistochemical markers were examined by Envision method.Eight patients were followed up.Results Of the 9 patients,8 had multiple subcutaneous nodules and plaques,which mainly involved the lower limbs in 8 patients and the trunk in 6 patients.Seven patients had fever.Three patients were subjected to the whole-body positron emission tomography-computed tomography (PET-CT),and 7 to bone marrow aspiration.No visceral tumors and hemophagocytic syndrome were found.Histopathological examination of skin lesions showed atypical mononuclear cells with large nuclei and deep staining,which mainly infiltrated the subcutaneous adipose tissue and were arranged in a circular pattern.Among 9 patients,infiltration of tumor cells was observed around skin appendages and blood vessels in the dermis in 5 patients.Immunohistochemical examination showed positive staining for βF1,CD3 and CD8 in tumor cells in 9 cases,positive staining for granzyme B and T-cell-restricted intracellular antigen-1 (TIA-1) in 8 cases,and negative staining for CD4,CD20,CD30 and CD56 in all the patients.Five patients received chemotherapy,including a child and a postpartum woman.One child received methylprednisoloue pulse therapy.During the follow-up,8 patients achieved a complete clinical remission after treatment.Conclusion SPTL is derived from α/β T cells,and histopathological and immunohistochemical examinations can be helpful for its diagnosis and differential diagnosis.

Objective To investigate the effects of bedside blinding method of active indwelling of nasojejunal tube combined with the nasogastric tube gastrointestinal decompression for patients with severe stroke.Methods 50 patients with severe stroke were selected and divided into two groups by using random number tables,which are the observation group and the control group,with 25 cases in each group.The patients in the observation group were treated with bedside blinding method of active indwelling of nasojejunal tube combined with the nasogastric tube gastrointestinal decompression,while the patients in the control group simply received bedside indwelling of nasogastric tube.The enteral nutritional goal -rate of target feeding volume on the 7th day and the 14th day after admission and trace the incidence of gastric stasis,the reflux and aspiration,the aspiration pneumonia in the patients of the two groups within 14 days and the situation of the days of mechanical ventilation,the days in ICU and the 30 -day mortality of patients were compared in the two groups.Results The enteral nutritional goal -rate of target feeding volume on the 7th day and the 14th day in the observation group were superior to those of the control group[The goal -rate of target feeding volume on the 7th day:88% vs.64%,χ2 =3.947,P =0.047;the goal -rate of target feeding volume on the 14th day:80% vs.52%,χ2 =4.367,P =0.037].Meanwhile the incidence of gastric stasis,the reflux and aspiration,the aspiration pneumonia in the patients of the observation group within 14 days were significantly lower than those in the patients of the control group within 14 days[The gastric retention rate:8% vs.56%(14 /25 ),χ2 =10.784,P =0.001;the reflux rate:0% vs.24%(6 /25),χ2 =4.735,P =0.03;the aspiration rate:8% vs.32%,χ2 =4.500,P =0.034;the incidence of aspiration pneumonia:24% vs.68%,χ2 =9.742,P =0.002].The days of mechanical ventilation and the days in ICU of the patients in the observation group are far less than those of the patients in the control group[The days of mechanical ventilation:(11.16 ±4.86)d vs.(13.72 ±3.67)d,t =-2.101,P =0.041;the days in ICU:(15.36 ±5.66)d vs.(18.72 ±2.99)d,t =-2.625,P =0.012].While there was no significant difference between the two groups on the 30 -day mortality(24% vs.32%,χ2 =0.397,P =0.529).Conclusion The bedside blinding method of active indwelling of nasojejunal tube combined with the nasogastric tube gastrointestinal decompression can significantly improve the enteral nutritional goal -rate of target feeding volume for patients with severe stroke and greatly reduce the incidence of gastric stasis,the reflux and aspiration,the aspiration pneumonia,and limit the days of mechanical ventilation and the days in ICU.Accordingly,it has the value of popularization in the clinical application.

Objective To investigate the protective effects of rhein lysinate ( RHL) on cardiac tissue damage in-duced by paraquat in experimental mice , and to clarify its mechanism .Methods In this study mice were assigned to the following three groups: control, paraquat model, and RHL-treated groups.The model of oxidative damage mice was established by intraperitoneal injection of paraquat .RHL-treated group was given RHL ( 50 mg/kg ) by gavage for one week before performing model .The other two groups were given equal volume of distilled water .For making model , paraquat was intraperitoneally injected in the paraquat model and RHL-treated group .The content of MDA was detected by thiobarbituric acid assay .The activities of SOD and GSH-Px were detected by biphenyl three phenolic autoxidation assay and NADPH coupling method respectivly .The pathological profile of cardiac tis-sue was observed by hematoxylin and eosin ( HE) staining and reactive oxygen species was observed by DCFH-DA staining .The change of proteins related to myocardial damage detected by Western blot .Results Compared with control group, the activities of SOD and GSH-Px decreased (P<0.05) and the content of MDA increased (P<0.05) in paraquat model group .However , these changes were attenuated byr RHL treatmen ( P<0.05 ) .The pathologi-cal examination indicated the structure of cardiac tissue was damaged and reactive oxygen species of cardiac tissue was increased after paraquat was given , however , these changes were attenuated after RHL treatmen .It was shown in western blot analysis that compared with control group , the expression of SIRT1 decreased, the acetylation of P53 and the expression of P 53 and P66 increased in paraquat-treated group .These changes were attenuated by RHL treatmen ( P<0.05 ) .Conclusions RHL may attenuate paraquat-induced cardiac injury in mice .

Objective:Nerve fibres distribution in the functional layer of endometrium of women with endometdosis was investigated.Methods:Histological sections of endometrial tissue were prepared from endometrialcurettings and hysterectomies performed on women with endometnosis(n=25)and without endometriosis(n=40).Immunohistochemistry was used to detect nerve fibres by highly specific polyclonal rabbit antibody PGP 9.5.The assessment of nerve fibre density was performed bv Image Pro Plus Discovery.Results:Nerve fibres were identified throughout the functional layers of the endometrium in all endometriosis patients,but not found in the functional layer of the endometrium in women without endometriosis(P<0.01).Conclusions:Nerve fibres detectad in the functional layer in all women with endometriosis may have important implications for understanding the generation of pain in these patients.

Objective To investigate the effects of rhein lysinate (RHL)on the expressions of TNF-α,IL-6 and NF-κB in the kidney tissue of senescence accelerated mouse prone 10 (SAMP 10)mice.Methods We selected 1 8 male mice (SAMP 1 0 )aged 7 months for the study and randomly divided them into blank control group and groups of different concentrations of RHL;six senescence accelerated mouse resistance 1 (SAMR 1 )served as the young control group.After 6 weeks’ treatment,HE staining was used to detect the pathological changes of the kidney.The expressions of TNF-α,IL-6 and NF-κB at the protein level were detected by immunohistochemistry and Western blotting.Results RHL treatment did not affect the body weight of SAMP 10 mice (P>0.05 ). Compared with SAMR 1 mice, contracted and destroyed renal glomeruli and infiltration of mononuclear macrophages were observed in control SAMP10 mice.However,this pathological process was blocked by RHL (25 mg/kg and 50 mg/kg ) treatments. In addition, the overexpressions of TNF-α, IL-6 and NF-κB and the phosphorylation of NF-κB in the kidney tissue of SAMP 10 mice could be inhibited by RHL treatments (P<0.05). Conclusion RHL inhibits the inflammatory reaction of the kidney tissue,which may be one of the mechanisms by which RHL exerts its kidney-protecting and anti-aging effects.

Objective To investigate influence of exogenous insulin in all-in-one parenteral nutrition on blood glucose in infants with very low birth weight (VLBW). Methods Forty-two infants with VLBWI admitted to the department of pediatrics of Xuzhou Hospital affiliated to Southeast University during September 2005 to March 2009 were randomly assigned to Group Ⅰ ( n = 13 ) with exogenous insulin added to all-in-one parenteral nutrition at infusion rate of 0.4 U·kg-1·h-1,GroupⅡ(n = 13) with exogenous insulin at infusion rate of 0.1U·kg-1·h-1 and Group Ⅲ (n = 16) with no exogenous insulin added.Their blood glucose was monitored every two hours. Chi-square test was used for comparing difference in blood glucose abnormality between the three groups and association between blood glucose levels at admission and during hospitalization was analyzed with Spearman correlation. Results Incidence of hyperglycemia and hypoglycemia was 10. 9 percent (29/265) and 18. 1 percent (48/265) in Group Ⅰ, 20. 8 percent (59/284) and 14. 1 percent (40/284) in Group Ⅱ , and 20. 5 percent (61/298) and 11.7 percent (35/298) in Group Ⅲ, respectively. There was significant difference in incidence of hyperglycemia between Groups Ⅰ and Ⅱ ( x2 = 9. 844, P = 0. 002 ) and between Groups Ⅰ and Ⅲ ( x2 = 9. 478, P = 0. 002 ), but no significant difference in it between Groups Ⅱ and Ⅲ ( x2 = 0. 008, P = 0. 928 ). There was significant difference in incidence of hypoglycemia between Groups Ⅰ and Ⅲ ( x2 = 4. 526, P =0. 033 ), but no significant difference in it between Groups Ⅰ and Ⅱ (x2 =1.653, P=0. 199) or between Groups Ⅱ and Ⅲ (x2 =0.709, P =0.400).No significant correlation between endogenous blood insulin level at admission and during hospitalization( r = 0. 082, P = 0. 661 ) was found. Conclusions Blood glucose in infants with VLBW can not be regulated timely by their endogenous insulin itseff. Exogenous insulin added to all-in-one parenteral nutrition at infusion rate of 0. 1 U · kg-1 · h-1 may not significantly reduce incidence of hyperglycemia,while incidence of hypoglycemia can be reduced by exogenous insulin at infusion rate of 0. 4 U · kg- 1 · h -1 that can increase incidence of hypoglycemia Therefore, exogenous insulin is not recommended to be prophylactically added to all-in-one parenteral nutrition for infants with VLBW.

OBJECTIVETo investigate the effect of functional blocking of endogenous miR-23a with a specific antisense oligonucleotide (ASO) on the proliferation and invasiveness of gastric adenocarcinoma cell line MGC803 in vitro.

METHODSA specific ASO targeting miR-23a, namely ASO-23a, was transfected into MGC803 cells to block endogenous miR-23a. The mRNA level of miR-23a in the transfected cells was detected with quantitative real-time PCR. The changes of cell proliferation following the transfection were detected with MTT assay and colony formation assay, and TUNEL assay and Transwell assay were employed to evaluate the changes in cell apoptosis and invasiveness, respectively.

RESULTSQuantitative real-time PCR demonstrated efficient functional blocking of endogenous miR-23a in MGC803 cells by ASO-23a. Suppression of miR-23a with ASO-23a obviously inhibited cell growth, colony formation and invasiveness of MGC803 cells and significantly enhanced the cell apoptosis.

CONCLUSIONASO-23a can efficiently block the function of endogenous miR-23a in MGC803 cells to inhibit cell proliferation and invasion and promote cell apoptosis.

Adenocarcinoma ; genetics ; pathology ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; MicroRNAs ; genetics ; Oligonucleotides, Antisense ; Stomach Neoplasms ; genetics ; pathology ; Transfection

Objective: To investigate the blood lead level of outpatient children and associated factors in Zhuzhou, and to offer targeted advice for the prevention and control of lead exposure. Methods: The lead level in blood of 1 600 children aged ≤14 years old and the content of serum calcium, iron, zinc, magnesium, copper were tested, and the children and their parents were given a questionnaire regarding influencing factors of blood lead exposure. Results: The average blood lead level of the children was (95.2±46.5)μg/L, The proportion of children with blood lead level ≥100 μg/L was 25.7%. The blood lead level between boys and girls had a statistical differences(Z=1.85, 2.85, P<0.05). The blood lead level was negatively correlated with serum calcium, iron, zinc, magnesium and copper(F=16.80，P<0.01). The risk factors for lead exposure included frequently drinking canned or bottled soft drinks, failing to wash one’s hands before meals, taking popcorn frequently, using coal for heat and for cooking, and constantly sucking fingers or biting fingernails (OR=2.12, 1.57, 1.46, 1.78, 3.24, P<0.01). Conclusion The blood lead levels of children in Zhuzhou is higher than national average level. We should strengthen environmental protection and behavioral interventions, and regularly monitor lead exposure among children.