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In order to explore the influence of purified donor regulatory T cells (Treg) infused after allogeneic bone marrow transplantation (allo-BMT) on GVHD and GVL effect in mice, an EL4 leukemia allo-BMT model of BALB/c-->C57BL/6 mice was established. The CD4(+)CD25(+)T cells were purified by positive selection using MACS. The recipients were injected with CD4(+)CD25(+)T cells or CD4(+)CD25(-)T cells within 4 hours respectively along with allo-BMT. Survival time, clinical GVHD score or histopathological features were observed after allo-BMT. The results showed that the mean survival time in leukemia control group was (17.9 +/- 0.7) days and all mice died of leukemia. The mean survival times in transplantation control group and CD4(+)CD25(-)T group were (23.2 +/- 1.6) and (22.3 +/- 1.9) days. Histopathological analysis in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD. The mean survival time in Treg group was (47.3 +/- 6.5) days. 70% of recipients were alive until day 60 without features of GVHD or tumor progression. Clinical GVHD scores in Treg group significantly decreased as compared to transplantation control group and CD4(+)CD25(-)T group (p < 0.05). It is concluded that allo-BMT combined with injection of donor CD4(+)CD25(+)Treg cells can efficiently prevent recipients from lethal GVHD with preserving GVL effect during allo-BMT.

Objective: To investigate the effects of early intensive therapy on beta cell function and long-term glycemic control in newly diagnosed type 2 diabetic patients with different recruiting fasting plasma glucose (FPG) levels., Methods: A total of 382 newly diagnosed type 2 diabetic patients with FPG 7.0 - 16.7 mmol/L were randomly assigned to therapy with insulin in the form of continuous subcutaneous insulin infusion (CSII) or multiple daily injection (MDI) or oral hypoglycemic agents (OHA, by using gliclazide and/or metformin) for initial rapid correction of hyperglycemia. The treatments were stopped after euglycemia had been maintained for 2 weeks. The patients were followed longitudinally on diet alone for 1 year. Intravenous glucose tolerances tests (IVGTTs) were performed and blood glucose, insulin and proinsulin were measured before and after therapy as well as at 1-year follow-up. Homeostasis model assessment (HOMA) of beta cell function and insulin resistance index (HOMA-beta and HOMA-IR) were calculated. All the patients were stratified on the recruiting FPG: stratum A (7.0 mmol/L

Versatile and novel reactions of 5-aminotetrazole with structurally diverse aryl aldehydes and building blocks with active methylene catalyzed by iodine were investigated in a multicomponent one-pot protocol. A series of 5,7-diaryl-4,7-dihydrotetrazolo[1,5-a]pyrimidines C and 5-methyl-7-aryl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylates D were obtained in moderate to good yields. Further exploration rapidly afforded various E in good to excellent yields; in addition, compound F was also obtained under the same condition. The structures of products were characterized by LC-MS, (1)H NMR, (13)C NMR, and elemental analysis.

Objective: To evaluate the diagnosis and surgical treatment of obstructive azoospermia., Methods: We analyzed the clinical data of 56 cases of obstructive azoospermia, 43 of them with ejaculatory duct obstruction (EDO), and the other 13 suspected of epididymal obstruction. The diagnostic methods included semen analyses, measurement of fructose and neutral alpha-glucosidase in the seminal plasma, transrectal ultrasonography (TRUS), and vasography when necessary. The 43 patients with EDO were treated by transurethral resection of the ejaculatory duct (TURED), and 11 of the 13 cases of suspected epididymal obstruction were confirmed by scrotal exploration and underwent either bilateral or unilateral vasoepididymostomy. The patients were followed up for 3 -51 months for postoperative semen quality and impregnation., Results: Of the 43 azoospermia patients with EDO treated by TURED, 36 (83.7%) showed improved semen parameters and 11 (25.6%) achieved pregnancies. Among the 11 cases of azoospermia with confirmed epididymal obstruction treated by vasoepididymostomy, 6 (54.5%) had sperm in the semen assay and 3 (27.3%) achieved pregnancies., Conclusion: Semen analyses, measurement of fructose and neutral alpha-glucosidase in the seminal plasma, TRUS and vasography are important diagnostic methods for obstructive azoospermia. TURED is effective for azoospermia with EDO, while vasoepididymostomy is preferable for the treatment of azoospermia with epididymal obstruction.

Objective: To determine the apparent n-octanol-water/buffer partition coefficient of andrographolide and dehydroandrographolide., Methods: The apparent n-octanol-water/buffer partition coefficients of andrographolide and dehydroandrographolide were measured by shaking flask method. The concentrations of andrographolide and dehydroandrographolide were analyzed by HPLC method., Results: The Papp of andrographolide and dehydroandrographolide were 3.90 (log Papp = 0.59) and 19.75 (log Papp = 1. 30) in water at 37 degrees C, respectively., Conclusions: The apparent n-octanol-buffer partition coefficients of andrographolide and dehydroandrographolide are influenced by pH, and the higher pH may decrease the apparent n-octanol-buffer partition coefficients of them. Andrographolide has the highest partition coefficient in pH6, and dehydroandrogapholide in pH5.

Objective: To prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression., Methods: The bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA., Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells., Conclusions: The genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Objective: To observe the cost-effectiveness of using continuous subcutaneous insulin infusion (CS II) and multi-point daily insulin injections (MDI) in controlling blood sugar in the newly hospitalized type 2 diabetes patients., Methods: Retrospective analysis on 86 cases taking CS II and 103 cases using MDI on a 'blood sugar control program' among the newly hospitalized patients with type 2 diabetes. The period for observation was 2 weeks, using cost-effectiveness analysis methods to evaluate the two treatment programs., Results: After two weeks of treatment, the effectiveness in the control of blood sugar in CS II group was similar to the MDI group, with no significant difference (P<0.05) and the adverse reactions were similar. Costs in the CS II program (Yuan/person) was less than in the MDI program (1478.34 vs. 1620.46), with significant differences (P< 0.05). The cost-effectiveness ratios (C/E) were 15.07 in the CS II group, and 16.34 in the MDI group, with no significant difference (P>0.05). In order to further reduce the cost of CS II group as a reference, the incremental cost-effectiveness ratio (DeltaC/DeltaE) of the MDI group was 129.20., Conclusion: Costs-effective of the CS II program was better than the MDI one in treating the newly hospitalized patients with type 2 diabetes, suggesting that CS II program might be a better choice for hospitals to carry on an intensive insulin therapy program.

The new phase-separation and immobilization technique known as fluorous biphase system (FBS) has become an active topic among researchers in both industry and academia. A series of fluorous biphasic reactions catalyzed by Lewis acid-type metal perfluorooctanesulfonates and metal bis(perfluorooctanesulfonyl)amides are reviewed in this paper. The recent development of fluorous phase transfer catalyst (PTC) is also discussed.

Objective: To investigate the changes in angiotensinogen (AGT), angiotensin II type 1 receptor (AT(1)R), endothelial nitric oxide synthase (eNOS) mRNA and protein expressions and nitric oxide (NO) content in the rat glomeruli in response to leptin stimulation., Methods: The glomeruli isolated from male SD rats were stimulated with 3 nmol/L leptin for 2 h. Real-time PCR and Western blotting were performed to analyze the mRNA and protein expressions of AGT, AT(1)R and eNOS in the glomeruli, and nitrite concentration in the glomeruli was measured by nitrate reductase assay., Results: In comparison with the control group, exposure to leptin increased the mRNA levels of AGT, ATR(1) and eNOS in the isolated glomeruli by 2.69-/+0.17, 3.77-/+0.16 and 2.56-/+0.29 folds (P=0.024, 0.018 and 0.044), and their protein levels by 2.06-/+0.10, 2.67-/+0.08 and 1.61-/+0.13 folds (P=0.021, 0.015 and 0.032), respectively. The NO production in the glomeruli was also increased by 2.77-/+0.14 folds (P=0.000) following leptin exposure., Conclusion: Leptin exposure of isolated rat glomeruli directly causes activation of the internal renal renin-angiotensin system and enhanced NO production, suggesting that leptin plays a role in the pathogenesis of maladaptation in renal hemodynamics in obesity.

Objective: To investigate the renal protective effects of sulodexide and its anti-oxidative stress mechanism in diabetic rats., Method: Thirty male SD rats were randomized into 3 equal groups, namely the control group, diabetic group, and sulodexide treatment group. Twelve weeks after establishment of rat diabetic models and administration of sulodexide, the rats were sacrificed for measurement of the urine volume, body mass, kidney mass/body weight ratio, plasma glucose, and glycosylated hemoglobin (HbA1c). Malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) activities in the renal tissue or serum were tested. Electron microscopy was performed to observe the pathological changes in the kidneys., Results: The urine volume, renal mass/body mass ratio, serum glucose, HbA1C, and serum and renal MDA levels all significantly increased in the diabetic rats in comparison with the normal controls (P<0.05). But the body weight and activities of SOD, CAT, and GSH-PX in the renal tissue in the normal control group were significantly higher than those in the diabetic and sulodexide group. After 12 weeks of sulodexide treatment, SOD, CAT, and GSH-PX activities in the renal tissue of rats were significantly increased in comparison with those in the diabetic rats (P<0.05). Electron microscopy showed obvious irregular thickening of the glomerular capillary basement membrane in the diabetic group with vacuolization in the mitochondria in the epithelial cells, and such pathological changes were significantly alleviated in the sulodexide treatment group., Conclusions: Sulodexide can effectively lower the urinary albumin excretion rate, improve the ultrastructural renal pathologies and prevent glomerular basement membrane thickening in diabetic rats, probably in association with the reduction of the MDA levels and enhancement of SOD, CAT, and GSH-PX activities.

Objective: To investigate the effect of allo-HSCT irradiation preconditioning on the homing of donor endothelial progenitor cells (EPCs)., Methods: The mononuclear cells were collected form mice bone morrows, then cultured in EGM-2 medium. The cells were identified after 5-7 days and were labeled with CFSE. The mice were divided randomly into four groups, control, EPCs transplantation, irradiation, and irradiation EPCs transplantation. The circulating ECs, circulating EPCs and CFSE positive cells in the peripheral blood, spleen and bone morrows of the mice were measured by FCS. The histological examination, electron microscope and immunofluorescence analyses of those tissues were also performed. The distribution of labeled EPCs was observed., Results: The cells were identified as CD45low, CD133+ CD31+, Dil-ac-LDL+. The number of those cells in the irradiation group increased gradually after preconditioning and reached peak at day 3 or 5 and sustained the peak level for 2 days, which was significantly different from the control (P < 0.05). Under the light microscopy and TEM, damaged endothelium was detected. In the irradiation and EPCs transplantation groups, CFSE labeled cells increased and reached peak at 18 hours, and then decreased till 72 hours. Green fluorescent cells were observed in different tissues at different times, and the strongest fluorescence intensity occurred at 36 hours., Conclusions: The EPCs can be induced from mice bone morrow mononuclear cells, with expressed phenotypic characterization. Irradiation in transplantation preconditioning can cause severe endothelium injury, which can start the mobilization of EPCs. The endothelium injury can induce extrinsic EPCs homing to the tissues which are badly injured. Endothelial progenitor cells

Objective: To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4+ CD25- T cells., Methods: The bicistronic lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected by lentiviral packing system, which including the packaging plasmid deltaNRF, the transfer plasmid and the envelope plasmid VSVG using liposome. Then manifested the efficiency of gene transduction with Flow Cytometry (FCM) and the expressions of Foxp3 mRNA and protein were assayed by RT-PCR and Western blotting., Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was constructed, the virus titres were above 10(6) IU/mL in the supernatant. After coculture with thus cells, the CD4+ CD25- T cells in experimental group expressed significantly higher amounts of Foxp3 mRNA and protein than control group, and the efficiency of gene transduction was about 55.95%., Conclusions: The self-inactivating lentiviral vector containing murine Foxp3 gene are successfully constructed. This gene delivery system can transduce Foxp3 gene into CD4+ CD25- T cells with highly efficient expression.

Objectives: To investigate the epidemiological and clinical characteristics of dyslipidemia as well as its treatment and influence on accompanying diseases in impaired glucose status among inpatients., Methods: A cross-sectional survey was conducted among the inpatients registered in ten university hospitals of Guangdong, China during the week before the Diabetes Day in 2004. The fasting blood glucose (FBG), lipid profiles, BMI, waist to hip ratio (WHR) and concomitant disorders of the first screen during the hospitalization period were recorded. Those who had FBG level from 5.6 to 6.9 mmol/L and not been previously diagnosed diabetes (PDM) underwent oral glucose tolerance test (OGTT)., Results: Of the 8753 inpatients investigated, 1067 cases had complete medical records (CMR case) including PDM cases and previously non-diagnosed diabetes ones with FBG > or = 5.6 mmol/L. Of the previously non-diagnosed diabetes cases with FBG levels from 5.6 to 6.9 mmol/L, 65.8% accepted OGTT. Of the CMR cases, 41.9% had PDM, 21.7% was newly diagnosed diabetes mellitus (NDM), 29.1% had impaired glucose regulation (IGR) and only 7.3% had normal glucose tolerance (NGT). The TG levels in NDM and PDM group were higher than those in IGR and NGT group (P < 0.05, respectively). The HDL-C levels in IGR, NDM and PDM group were lower than those in NGT group (P < 0.05, respectively). Sixty-nine point six percent of the diabetes mellitus (DM) inpatients was accompanied with dyslipidemia and the rate was higher than those in NGT (56.4%) and IGR inpatients (52.5%, P < 0.05, respectively). Only 22.8% of the PDM inpatients underwent treatment of dyslipidaemia and just 3.4% achieved the target suggested by the guideline of ATP-III. BMI was higher and waistline longer in the PDM and NDM inpatients than those in the NGT cases (P < 0.05, respectively). Seventy-two point eight percent of the PDM inpatients was complicated with more than one type of vascular diseases. Nine point seven percent and 0.2% of the NDM inpatients were tormented by diabetic nephropathy and diabetic retinopathy respectively., Conclusions: More inpatients with accompany DM or IGR had concomitant dyslipidemia than those with NGT, which included hypertriglyceridemia, hypo-high-density lipoproteinemia and metabolic syndrome. Concomitant vascular diseases were more frequently found in PDM inpatients than in the others. Some of the NDM and IGT inpatients were complicated with microvascular diseases.

Objective: To construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene., Methods: Lentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay., Results: The MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%., Conclusions: The lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Background: Diabetes mellitus has become epidemic in recent years in China. We investigated the prevalence of hyperglycaemia and inadequate glycaemic control among type 2 diabetic inpatients from ten university teaching hospitals in Guangdong Province, China., Methods: Inadequate glycaemic control in diabetic patients was defined as HbA1c = 6.5%. Therapeutic regimens included no-intervention, lifestyle only, oral antiglycemic agents (OA), insulin plus OA (insulin + OA), or insulin only. Antidiabetic managements included monotherapy, double therapy, triple or quadruple therapy., Results: Among 493 diabetic inpatients with known history, 75% had HbA1c = 6.5%. Inadequate glucose control rates were more frequently seen in patients on insulin + OA regimen (97%) than on OA regimen (71%) (P < 0.001), and more frequent in patients on combination therapy (81% - 96%) than monotherapy (75%) (P < 0.05). Patients on insulin differed significantly from patients on OA by mean HbA1c, glycemic control rate, diabetes duration, microvascular complications, and BMI (P < 0.01)., Conclusions: This study showed that glycaemic control of type 2 diabetic patients deteriorated for patients who received insulin and initiation time of insulin was usually delayed. It is up to clinicians to move from the traditional stepwise therapy to a more active and early combination antidiabetic therapy to provide better glucose control.

Objective: To explore the silencing effect of short hairpin RNA (shRNA) on the CD28 of mice T lymphocytes by CD28-shRNA expressing plasmid evaluate the interfering effects (chronology and stability) mediated by shRNA and select out the most efficient CD28 shRNA sequence., Methods: Three CD28 specific and one non-specific shRNA expressing plasmids were constructed and then transfected separately into mice spleen T lymphocytes. Non-transfected cells and non-specific shRNA were taken as controls. Inhibitory effect of CD28 shRNA was demonstrated by real-time quantitative PCR and Western blots. The sequence of the highest RNA interference (RNAi) efficacy was screened., Results: (1) CD28 shRNA expressing plasmids were successfully constructed; (2) Three CD28 specific shRNAs effectively inhibited the expression of CD28 at the mRNA and protein levels, and there was a statistically significant difference comparing with the controls (P < 0.01): The copies of CD28 in mice spleen cells at the mRNA levels were persistently decreased by 99.62%, 99.89% and 99.80% respectively after 20 days, and so did at the protein level [(84.90 +/- 0.65)%, (96.49 +/- 0.03)%, (91.76 +/- 0.32)% respectively]. The highest inhibitory rate was in CD28 shRNA-2 group., Conclusions: (1) Specific shRNA can mediate long-term and stable silencing effects on CD28 gene; (2) shRNAs matching different sites of CD28 gene exert differential inhibitory effects.

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.

Objective: To compare the effect of continuous and discontinuous density gradient centrifugation for purification of human pancreatic islets with COBE 2991 cell processor., Methods: Human pancreases were obtained from brain-dead donors and stored in cold UW solution. The connective tissues were removed from the pancreases, and the pancreatic ducts were perfused with a cold enzyme (Liberase). The islets were then separated by gentle mechanical dissociation and purified with discontinuous (10 pancreases) or continuous (8 pancreases) gradients of HCA-Ficoll in COBE 2991 cell processor. Samples were collected in duplicate for determination of the quantity of islets, islet equivalents (IEQ), and the purity., Results: The weights of the pancreases before and after connective tissue removal and pancreas duct perfusion, and the quantity of islets obtained (including islets quantity of different diameters and total IEQ) after dissociation were not significantly different. Continuous gradient of HCA-Ficoll, compared with discontinuous gradient, resulted in significantly greater final islet quantity (55,000 IEQ vs 206,000 IEQ, P=0.000) and islet purity (58.0%-/+8.0% vs 33.5%-/+10.3%, P=0.000) and also greater number of islets with a diameter lager than 200 microm (P<0.01)., Conclusion: Continuous density gradient centrifugation can be more effective than discontinuous gradient in islet purification.

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.

Objective: To establish an semi-automated effective method for large-scale purification of islet cells from human pancreas., Methods: Human pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro., Results: The number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001)., Conclusion: The established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.

Objective: To observe the therapeutic effect of warming needle moxibustion on prolapse of lumbar intervertebral disc., Methods: Seventy-eight cases were randomly divided into a test group and a control group, 39 cases in each group. The control group were treated with acupuncture at Shenshu (BIL 23), Mingmen (GV 4), Yaoyangguan (GV 3) and Weizhong (BL, 40), etc. , and the test group with acupuncture at the same acupoints as in the control group plus warming needle moxibustion at Mingmen (GV 4), Yaoyangguan (GV 3) and Weizhong (BL 40)., Results: The total effective rate of 94.9% in the test group was significantly better than 71.8% in the control group (P < 0.01). The incidence rate of needling sensation propagating along the channel (84.6%) in the test group was significantly higher than 56.4% in the control group (P < 0.01)., Conclusion: The warming needle moxibustion stimulating the needling sensation propagating along the channel has a better therapeutic effect on prolapse of lumbar intervertebral disc.

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.

Objective: To investigate the characteristics of the dysfunction of islet beta-cell in newly diagnosed type 2 diabetic patients., Methods: Intravenous glucose tolerance test (IVGTT) was carried out on 352 newly diagnosed type 2 diabetic patients and 48 subjects with normal glucose tolerance (NGT) and then blood samples were collected 1, 2, 4, 6, and 10 minutes later to measure the plasma glucose and insulin to calculate the acute insulin response (AIR) and the area under the curve of insulin (AUC of insulin), homeostasis model assessment beta-cell (Homabeta), and Homa IR (insulin resistance)., Results: The median AIR of the type 2 diabetic patients was -33.7 pmol/L, significantly lower than that of the NGT subjects (6962.0 pmol/L, P < 0.001). The median AUC of the type 2 diabetic patients was 834.2 pmol/L, significantly lower than that of the NGT subjects (7934.7 pmol/L, P < 0.001). When the fasting plasma glucose (FPG) of the type 2 diabetic patients was above 7.0 mmol/L, the AIR value was remarkably reduced with a median level of 317.3 pmol/L, and then subsequently disappeared when the FPG was above 9.0 mmol/L. After adjustment of the insulin resistance assessed by HOMA IR, the Homabeta of the type 2 diabetic patients was reduced to be 30% that of the NGT subjects (3.7 +/- 0.9 vs 5.9 +/- 0.9, P < 0.001). Both the fasting proinsulin concentration and the ratio of fasting proinsulin to fasting insulin of the type 2 diabetic patients were significantly higher those of the NGT subjects (22.6 pmol/L +/- 14.7 pmol/L vs 11.5 pmol/L +/- 7.1 pmol/L, P < 0.001; and 30.1% +/- 20.5% vs. 12.1% +/- 9.6%, P < 0.001)., Conclusion: The dysfunction of islet beta-cell in newly diagnosed type 2 diabetic patients is mainly represented by the disappearance of AIR and the evident decline of AUC and HOMA B, and the decrease of quality of insulin secretion.

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.

Objective: T o determine the quality standard of Yixin Tablet., Methods: The nature differentiation of Radix Ginseng, Radix Astragali, Rhizoma Chuanxiong, Fructus Schisandrae was determined by TLC and content of salvianolic acid B in Yixin Tablet was determined by HPLC., Results: The nature differentiation separate degree is kind and special attribute was strong. The compounds were base-isolated on the column of C18 which gradient eluted with acetonitrile and H2O (0.05% H3PO4) and detective wavelength was 280 nm. The linearity was obtained over the range of 0.12504-2.50080 microg (r = 0.9999) and the average recovery rate was 99.35%., Conclusion: The method is simple, convenient and accurate. The sensitivity is high. It can be used in the quality control of Yixin Tablet.

Objective: To develop a method for quantitative assay of alpha-Gal on pig cell surface., Methods: After centrifugation of the anticoagulated blood samples, the white blood cell layer was collected for addition of FITC-BSIB4 and analysis using FCM as well as Laser Scan Confocal Microscope (Bio-Rad)., Results: With human blood group B as negative control, and mouse myeloma SP2/0 cells as positive control. the peripheral blood cells of two Chinese pigs were all positive, there were about 9.16 x 10(5) alpha-Galactosyl epitopes expressed on the cell-membrane of granulocytes 1.37 x 10(5) on the monocytes of Banna Minipig Inbred Line (BMI), and 1.16 x 10(6) on the granulocyte and 2.45 x 10(5) on the monocytes of Sichuan White Pig (SWP). The levels of alpha-Galactosyl expression on the surface of blood cells in BMI and SWP differed in various cells, the sequence from high to lower expression levels being on the platelets, granulocytes, monocytes, erthrocytes in each species of pig., Conclusion: The method is useful for quantitative assay of alpha-Gal on pig cell surface, and the assay may be used for alpha-Gal for on-site large-scaled screening of living pigs.

This study sought to investigate whether mesenchymal stem cells (MSC) derived from Banna Minipig Inbred-line (BMI-MSC) suppressed human peripheral blood lymphocyte (hPBLs) proliferation in a one-way mixed lymphocyte reaction system. BMI-MSC failed to stimulate proliferative responses by hPBLs, which were activated by allogenic endothelial cells, BMI-PBLs and non-specific mitogenic stimuli. Furthermore, BMI-MSC also suppressed proliferation of hPBLs stimulated by mismatched allogenic, as well as xenogenic PBLs, and the mitogenic stimulus ConA. The suppression occurred in dose-dependent fashion when the ratio of hPBLs to BMI-MSC varied from 1 to 5 fold; fewer, BMI-MSC (0.001 to 0.01 times) showed no obvious suppression. When BMI-MSC were added to hPBLs stimulated for 72 hours, the proliferative suppression was still evident. Addition of anti-FasL or anti-TGF-beta1 antibody attenuated the proliferative suppression, while antibody against IL-10 had no effect on it. Further immunofluorescence analysis demonstrated that FasL and TGF-beta1 constitutively expressed BMI-MSC. These findings suggest that BMI-MSC suppress hPBLs proliferation relying on FasL and TGF-beta1 mediated pathways.