Formate dehydrogenase(FDH)coding gene was amplified from genomic DNA of Pichia pastoris by polymerase chain reaction, and the codon TAG(bases 649-651)was mutated to GAG using site-directed mutagenesis.The recombinant plasmid pET-FDH was con- structed by inserting the mutated DNA fragment into expression vector pET-22b(+),and transformed into E.coli BL21(DE3).FDH was expressed as a form of soluble prutein fused with 6?His tag at high level through IPTG induction.The amount of FDH was up to about 30% of the total cell protein.The cells-free crude extract was purified by one affinity chromatographic step,and resulting enzyme preparation revealed a specific activity of 6.45 U/mg.

Objective To study the correlation between chronic gastritis with benign mucosal nodular-change,Helicobacter pylori(H.pylori)infection and lymphoid follicles infiltration.Methods During July 1,2004 to June 30,2005 patients with chronic gastritis and benign mucosal nodular-change were identified by chromo-endoscopy with anabrosis indigo carmine staining at the antrum.Multiple biopsies were obtained for H.pylori detection with quick test and for pathology examination of mucosal lymphoid follicles formation and lymphocyte infiltration,as well as H.pylori infection.Results The patients were divided into nodular gastritis group,atrophy gastritis group and verrucous gastritis group with mean age of(31.00?11.62),(58.61?12.14)and(51.29?12.99)years old,respectively.The patients with nodular gastritis were the youngest(P

0.05).The off-pump group had a shorter respiratory support time [(4.8?1.9)h vs(8.9?2.1)h,t=17.453,P=0.000],less drainage volume[(390?152)ml vs(660?111)ml,t=17.173,P=0.000],and required less blood transfusion [(270?77)ml vs(510?144)ml,t=17.861,P=0.000] than the on-pump group.In the off-pump group,3(2.0%)of the 150 patients developed renal function injury that is significantly more than that in the on-pump group [11/140(7.9%),?2=5.407,P=0.020].Conclusions Off-pump CABG is as effective as the on-pump surgery;moreover,it is a better choice for patients with high-risk coronary disease since this technique is superior in reducing respiratory support time,volumes of chest drainage and blood transfusion,and renal function injury.

Objective To review the strategies for multiple coronary artery bypass in patients with calcified ascending aorta.Methods From June 2002 to December 2007,36 patients with multiple coronary artery disease and calcified ascending aorta underwent OPCAB without aortic side-bite clamping.The patients(28 males and 8 females) aged from 60 to 83 years with a mean of(69.1?6.7) years.Fifteen of the patients had a history of stroke.Coronary angiogram revealed that 3 patients had double vessels disease and the others had triple vessels disease,involving the left main stem in 14 cases.The left ventricular ejection fraction ranged from 0.32-0.69.Calcified ascending aortic plaque can be explored in 30 of the patients during operation while intra-operative transesophageal echocardiography indicated calcified aorta only in 28.No-touch technique was used in 22 patients either with LIMA as the only inflow and the saphenous vein grafts was anastomosed to the LIMA in 15 patients,or with bilateral internal mammary arteries as the only inflows in 7 patients.Proximal anastomotic device was employed in 14 patients to finish the proximal anastomosis of greater saphenous vein grafts without aortic side-biting clamping.Intra-operative graft flow meter was used to check the flow of grafts before chest closure. Results Totally 116 cases of distal anastomosis were performed in the 36 patients(2-5 in each,mean 3.2?0.9).The patients woke up from anesthesia in 2 to 22 hours.Intraoperative measuring of graft flow revealed satisfying blood flow in all the grafts.In the 15 patients with LIMA as the only inflow,the total graft flow in the main stem of LIMA was basically the sum of each connected grafts.Only one patient died after surgery,showing a mortality rate of 2.7%.Eight patients(26.7%) had transient atrial fibrillation after the operation,2 showed pulmonary infection,4 had chest fluid,1 showed infection of the incision at the lower limb.All the complications were cured.30 patients achieved a 6-to 60-month follow-up with a mean of(33.8?11.2) months.During the period,11 patients(37 grafts) were examined by angiography,which showed that all the grafts of LIMA to LAD and vein grafts were patent,except in two vein grafts,which were occluded in the diagnol branches. Conclusions In patients with remarkable ascending aortic calcification,OPCAB combined with aorta no-touch technique or proximal anastomosis device can effectively decrease the incidence of postoperative stroke.

BACKGROUND:Mesenchymal stem cells(MSCs) have immunoregulation,can reduce the occurrence rate of graft versus host disease following transplantation,and treat autoimmune disease. OBJECTIVE:To observe the immunoregulation of human umbilical cord(UC) -MSCs on bone marrow T-lymphocyte of patients with aplastic anemia. DESIGN,TIME AND SETTING:A randomized grouping design,in vitro cytological controlled study. Cases were obtained from the Department of Hematology,First Hospital and Second Hospital Affiliated to Nanchang University. Experiments were conducted at the Institute of Hematology,Second Hospital Affiliated to Nanchang University from September 2007 to November 2008. PARTICIPANTS/MATERIALS:A total of 22 patients with aplastic anemia were enrolled at the Department of Hematology,First Hospital and Second Hospital Affiliated to Nanchang University. There were 6 cases of severe aplastic anemia(3 males and 3 females) ,with a median age of 38(16-68) years;16 cases of chronic aplastic anemia(9 males and 7 females) ,with a median age of 41(25-59) years. Umbilical cord was obtained from healthy full-term fetus by normal uterine-incision delivery for isolation,culture and determination of human UC-MSCs. METHODS:MSCs were extracted from human umbilical cord. T-lymphocytes were isolated from bone marrow of patients with aplastic anemia by density gradient centrifugation. MSCs were cocultured with T-lymphocytes. In the control group,T-lymphocytes were incubated at 1?105/well. In the UC-MSCs group,human UC-MSCs were incubated at various densities of 1?105/well,0.5?105/well,0.1?105/well and 0.05?105/well. In the experimental group,T-lymphocytes were cocultured with UC-MSCs at 1:1,1:0.5,1:0.1,1:0.05. MAIN OUTCOME MEASURES:Inhibitory rate of human UC-MSCs at various concentrations on T-lymphocytes of aplastic anemia patients was measured by MTT assay. CD4+/CD8+ changes of the T cells were analyzed by flow cytometry. Expression of the two marrow hematopoietic factors(interleukin-2 and ?-interferon) in the supernatant was evaluated by ELISA. RESULTS:The second passage of MSCs had typical morphology of fibroblast,and highly expressed CD166,CD29,CD54,but lowly expressed CD13,CD34,CD45. MSCs could be induced into adipocytes. UC-MSCs showed inhibitory effect on lymphocyte proliferation of aplastic anemia patients when UC-MSCs and lymphocytes mixed at the ratio of 1:1,0.5:1,0.1:1,and their inhibitory rate were(56.2?12.1) %,(43.7?10.4) %,(28.6?8.9) %. The effect was the strongest at the ratio of 1:1. UC-MSCs adjusted the proportion of CD4+ and CD8+,inhibited the secretion of interleukin-2 and ?-interferon by T cells of aplastic anemia patients. CONCLUSION:Human UC-MSCs can mediate an immunoregulation effect on T lymphocytes of aplastic anemia patients in vitro and the inhibitory effects are dependent on the amount of UC-MSCs.

Hepacivirus ; Hepatitis C ; complications ; epidemiology ; Humans ; Lichen Planus, Oral ; complications ; epidemiology ; virology

Objective:To explore the clinical efficacy, especially the comprehensive improvement of blood glucose and lipid of probucol and metformin in the treatment of type 2 diabetes mellitus with hyperlipidemia. Methods:Totally 105 patients with type 2 di-abetes mellitus complicated with hyperlipidemia were randomly divided into the control group1 (35 cases), the control group 2 (34 ca-ses) and the observation group (36 cases). The control group 1 was treated with diet control, exercise and metformin, the control group 2 was treated with rosuvastatin calclum tablets based on the group 1, and the observation group was treated with probucol based on the group 1. The three groups were continuously treated for 12 weeks. The improvement of the fasting blood-glucose (FPG), 2h postprandial blood glucose (PBG), hemoglobin A1C (HbA1C), fasting insulin levels (Fins), insulin resistance indices (HOMA-IR) and TC, TG and HDL-C, and the adverse reactions among the three groups were compared. Results:The total effective rate in the ob-servation group was higher than that in the control group 1 and 2 (P<0. 05). After the treatment, all the indices of blood glucose and lipid in the three groups were significantly improved (P<0. 05), and those of blood glucose in the observation were better than those in the control group 1 and 2(P<0. 05), those of blood lipid in the observation group and the control group 2 were better than those in the control group 1 (P<0. 05), and the level of HDL-C in the observation group was much higher than that in the control group 2 (P<0. 05). The adverse drug reactions in the three groups were mild without statistical significance (P>0. 05). Conclusion:Probucol as one of lipid-lowing drugs with antioxidant action combined with metformin can improve blood lipid and lower blood glucose at the same time, which is worthy of promoted application in clinics.

Objective To investigate the characteristics of HBsAg and HBcAg in combination pulsed monocyte-derived DCs(MoDC) derived from patients with HBV-associated hepatocellular carcinoma(HCC).Methods MoDCs were generated from 20 HBV-associated HCC patients,and pulsed with recombinant human serum albumin(rHSA)as conDC(control DC),or pulsed with HBsAg andHBcAg in combination as scDC.Phenotypic patterns of MoDCs were characterized by flow cytometry,and the levels of cytokines releasedby MoDCs were analyzed by ELISA,and frequencies of IFN-γ-producing antigen-specific autologous T cells were measured by Elispotassay.HBV-specific CD8 T cells proliferation was determined by pentamer staining.Results 1.The levels of MHC and costimulatorymolecules expressed on scDC were significantly higher than those on immature MoDC(imaDC)in two groups,and the levels of MHC andcostimulatory molecules expressed on imaDC,conDC and scDC in HBsAg+,anti-HBe+,anti-HBc+patients(1-4-5 positive group)were significantly higher than those in HBsAg+,anti-HBc+patients(1-5 positive group);2.The levels of IL-12 and IL-10 produced byscDC were higher than those produced by imaDC and conDC in both groups,and the levels of IL-12 produced by imaDC,conDC and scDCin 1-4-5 positive group were higher than those in 1-5 positive group.3.The frequencies of IFN-γ-producing T cells induced by scDC werehigher than those by conDC in 1-4-5 positive group.4.scDCs from 4 cases of HLA-A2+patients in 1-4-5 positive group could induceautologous T cells to generate HBVcore18-27-specific CD8 T cells.Condusions HBsAg and HBcAg pulse in combination couldsubstantially reverse the impaired function of MoDCs in HBv-associated HCC patients,and boost MoDC to induce HBV-specific T cellsresponse,especially in HBsAg+,anti-HBe+ and anti-HBe+ patients.

Objective To study the expression of human kallikrein gene(KLK) 4 and KLK5 in ovarian cancers,and to investigate the pathogenesis in malignant tumors. Methods Fifty specimens of ovarian cancers were divided into three groups: malignant tumor group(n=23),borderline tumor group(n=6) and control group(normal or benign tumor,n=21).Fluorescent quantitative RT-PCR was employed to determine the expression of KLK4 and KLK5 in these specimens. Results The expression of KLK4 in ovarian cancers was significantly higher than that of the control group(P

Objective To explore the effects of early growth response gene-1 (Egr-1) on bone marrow mesenchymal stem cells (BMSC) proliferation and osteogenic differentiation,which is aimed at providing new molecular targets for the treatment of osteoporosis.Methods Bone marrow was collected from adult men and the BMSCs were cultured primarily and observed by microscope.Meanwhile,flow cytometry was used for BMSCs phenotypic identification;After transfection of pcDNA3.1/Egr-1 into BM SCs,the level of BMSCs proliferation was determined by MTT respectively on the 2 d,4 d and 6 d;On the 7 d after transfection,the ALP activity assay was used for testing the ALP activity in BMSCs.And then,alizarin red S-calcium kit was used for measuring the calcified knots respectively on the 7 d,14 d and 21 d;On the 21 d after transfection,real-time qPCR and Western blotting were used respectively for measuring the expression of mRNA and protein of Egr-1,Runx2 and NDRG1;Further,BMSCs were transfected with Egr-1 siRNA,and the content of calcium nodules,ALP activity,the expression of Egr-1,Runx2 and NDRG1 were detected as above methods.Results The cells cultured in vitro showed high level of CD90 and CD29 and very low level of CD34 and CD45,which is accorded with the characteristic of BMSCs.The pcDNA3.1/Egr-1 transfection for BMSCs had no effect on cells prolifera tion.However,the calcified knots,ALP activity and the expression of Egr 1,Runx2 and NDRG1 were increased after transfection of pcDNA3.1/Egr-1 for BMSCs.In addition,Egr-1 siRNA showed the opposite effect with pcDNA3.1/Egr-1 transfection for BMSCs.Conclusion Egr-1 induces osteogenic differentiation of BMSCs by promoting NDRG1 but has no effects on proliferation of BMSCs.