This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.
Acetylcholinesterase ; genetics ; metabolism ; Cholinesterase Inhibitors ; analysis ; pharmacology ; HEK293 Cells ; Humans ; Indans ; analysis ; pharmacology ; Inhibitory Concentration 50 ; Kinetics ; Piperidines ; analysis ; pharmacology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo explore the effects of hippophae juice on free radical metabolism of rat skeletal muscle and partial biomarkers in blood.

METHODSRandomly dividing the 30 SD rats into 3 groups (n = 10): sedentary group, training group and hippophae training group. Measuring related indices of skeletal muscle and blood in rat after 6 week training and hippophae juice supplement.

RESULTSCompared with training group, hippophae training group showed obviously longer exhaustive time, significantly increased antioxidant enzyme in skeletal muscle, remarkably decreased malonaldehyde (MDA) content in skeletal muscle, obviously increased testosterone (T) and hemoglobin (Hb) content in blood, significantly decreased creatine kinase (CK).

CONCLUSIONHippophae juice can impove the antioxidant ability of rat skeletal muscle, the level of T and Hb in blood, delay fatigue, therefore effectively enhance the aerobic stamina of rat.

Animals ; Creatine Kinase ; blood ; Free Radicals ; metabolism ; Hemoglobins ; metabolism ; Hippophae ; Male ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood

Objective To observe the effects of 75 gram glucose oral tolerance test (75 g OGTT) and standard mixed meal test (SMMT) on insulin secretion function of the islets of Langerhans and plasma free fatty acid (FFA) in patients with type 2 diabetes mellitus.Methods Seventy-six patients with type 2 diabetes without using insulin and with no obvious complications were recruited for 75 g OGTT following overnight fasting on the first day and SMMT (bread 50 g,egg 50 g and milk 250 ml) on the 7th day.Blood specimens were collected from each patients before the tests and 30 min,60 min,120 min and 180 min after glucose or meal load to measure their levels of plasma glucose,serum insulin,C peptide,FFA and lipids (total cholesterol,triglyceride,high-density and low-density lipoprotein cholesterol).Results No difference in fasting plasma glucose,serum insulin,C peptide,FFA and lipids between 75 g OGTT and SMMT was found.Postprandial plasma glucose 30 min,60 min,120 min and 180 min after 75 g OGTT was significantly higher than that after SMMT,with (15.3?3.5) vs (9.9?3.4) mmol/L,(18.2?4.8) vs (12.8?4.0) mmol/L,(16.3?5.8) vs (12.2?4.9) mmol/L and (10.6?5.4) vs (9.5?4.5) mmol/L (F=28.1,P

BACKGROUNDThe B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.

METHODSAccording to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.

RESULTSThree siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05).

CONCLUSIONSThree different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent ; Adult ; CD28 Antigens ; genetics ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Gene Silencing ; Humans ; Lymphocytes ; metabolism ; RNA, Small Interfering ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

OBJECTIVETo screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).

METHODSBased on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.

RESULTCo-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.

CONCLUSIONThe expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

Base Sequence ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Embryo, Mammalian ; Green Fluorescent Proteins ; genetics ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B e Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Kidney ; cytology ; Liver Neoplasms ; pathology ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; RNA, Small Interfering ; RNA, Transfer, Val ; genetics ; RNA, Viral ; genetics ; Transfection

The aim of study was to investigate the inhibitory effect of small interfering RNA on evi1 gene expression and biological characteristics in HEL cells and its mechanism. 3 siRNA (siRNA-1, siRNA-2, siRNA-3) specific for evi1 gene were synthesized and transfected into HEL cells in vitro. Experiments were divided into test and control groups. MTT method was used to assay the inhibitory effect of siRNA on cell proliferation; semiquantitative RT-PCR was used to detect the expression of evi1 gene mRNA; the cell viability was determined by trypan blue dye test; the change of cell cycle and apoptosis of cells were analyzed by flow cytometry. The results showed that siRNA-1 had strongest effect, and inhibitory effect was most obvious at 48 hours after transfection. When the concentration of siRNA raised to 120 nmol/L, the inhibitory rate reached to the peak. The inhibitory rate of siRNA-1 on proliferation of HEL cells, relative expression level of evi1 mRNA and cell viability at 48 hours after transfection were 72.22 ± 2.80%, 27.31 ± 1.11% and 26.05 ± 2.49%, which had significant difference from other groups (p < 0.001). The siRNA resulted in arrest of cell cycle at G(0)/G(1) phase, the cell amount at S phase obviously decreased, the apoptotic rate of HEL cells obviously increased (p < 0.01). It is concluded that the siRNA specific for evi1 gene can suppress the proliferation of HEL cells, reduce the expression of evi1 mRNA, decrease the cell viability, arrest the cell cycle at G(0)/G(1) phase, suppress cell mitosis, and promote cell apoptosis.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA-Binding Proteins ; metabolism ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; pathology ; MDS1 and EVI1 Complex Locus Protein ; Proto-Oncogenes ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transcription Factors ; metabolism

The objective of this study was to investigate the specificity of detecting the phosphotyrosine level with anti-phosphotyrosine monoclonal antibody PY20 for diagnosis and prognosis of patients with chronic myeloid leukemia (CML) and the possibility of its clinical application. The positive rate of PY20 in 28 newly diagnosed CML patients was detected by flow cytometry using anti-PY20 antibody, the bcr-abl fusion gene was detected by nested RT-PCR, the Ph chromosome was measured by R-banding cytogenetic analysis, and the coincidence of PY20 positive rate with results of bcr-abl fusion gene and Ph chromosome detection was compared. In addition, the positive rate of PY20, the changes of bcr-abl fusion gene and Ph chromosome were determined in follow up 7 CML patients after allo-hematopoietic stem cell transplantation. The results indicated that the positive rates of PY20 in 28 newly diagnosed CML patients in groups of chronic phase (CP), accelerated phase (AP), and blast phase (BP) were (40.31% +/- 1.22)%, (77.28 +/- 1.14)% and (78.12 +/- 1.32)% respectively. The positive rate of PY20 in CP was lower than that in AP and BP (p < 0.05). There was no difference in positive rate of PY20 between AP and BP (p > 0.05). PY20 expression level of leukocytes from peripheral blood and bone marrow showed no difference (p < 0.05). The positive rates of PY20 in patients with CR, PR and NR were (15.56% +/- 1.51)%, (38.73% +/- 2.31)% and (60.43% +/- 2.04)% respectively. The positive and negative coincidence between PY20 and RT-PCR was 92.31% and 95.45% respectively. The positive and negative coincidence between PY20 and Ph Chromosome in newly diagnosed patients was 88.46% and 95.46% respectively. Ph chromosome and PY20 were all negative in 7 CML patients after allo-HSCT. Bcr-abl fusion gene was negative persistently in 5 patients, but in the other 2 patients, the fusion gene was persistently positive. In conclusion, the detection of the level of phosphotyrosine in CML cells has high sensitivity and specificity. The results of PY20 cell positive rate combined with detection of bcr/abl fusion gene and Ph chromosome might be useful in diagnosis as a good index of monitoring.
Adolescent ; Adult ; Antibodies, Monoclonal ; chemistry ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Male ; Middle Aged ; Philadelphia Chromosome ; Phosphotyrosine ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Young Adult

To establish a quantitative assay for telomerase activity and analyze the telomerase activity in peripheral blood mononuclear cells (PBMNC) from patients with acute leukemia, a fluorescent dye, PicoGreen, was added to the products after telomere repeat amplification protocol. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Telomerase activity was detected in PBMNCs from 20 cases of normal individuals and 25 patients with acute leukemia. The results showed that the fluorescence of PicoGreen binding to double-stranded DNA specifically was enhanced with increase of DNA quantities. In conclusion, the met hod is rapid, simple and quantitative, the telomerase activities of PBMNCs from acute leukemia patients are significantly higher than that of the normal controls.
Acute Disease ; Adolescent ; Adult ; Aged ; Cell Line ; DNA, Neoplasm ; genetics ; metabolism ; Female ; Fluorescent Dyes ; chemistry ; Humans ; Leukemia ; blood ; enzymology ; genetics ; Leukocytes, Mononuclear ; enzymology ; Male ; Middle Aged ; Organic Chemicals ; Telomerase ; chemistry ; genetics ; metabolism

OBJECTIVETo explore the influence of the lentiviral vector mediated mouse genetic engineering regulatory T cells (Treg) infusion on post-allogeneic bone marrow transplantation (allo-BMT) acute graft-versus-host disease (GVHD) in mice.

METHODSLentivirus-mediated Forkhead box P3 (Foxp3) gene was transduced into BALB/c mice CD4(+)CD25(-) T cells (Treg) to construct engineered Tregs in vitro. An allo-BMT model of BALB/c to C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells plus the genetic engineering Tregs. Survival time, clinical GVHD score, histopathological findings, activation of donor T cells or serum levels of inflammatory cytokines were observed after allo-BMT.

RESULTSThe mean survival times for radiation alone group (Gp I), transplantation control group (Gp II), engineering Treg infusion group (Gp III) and empty vector control group (Gp IV) were (8.8 ± 0.6) d, (36.7 ± 2.5) d, (51.6 ± 4.0) d and (34.1 ± 2.3) d, respectively. The survival time was significantly longer in Gp III than in other groups (P < 0.05). Histopathological finding in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in Gp II and Gp IV, while no histological signs of GVHD were observed in long survival recipients in Gp III, and clinical GVHD scores in Gp III were significantly lower than that in Gps II and IV. The numbers of donor T cells and the percentage of IFN-producing donor T cells in the spleen of recipients in Gp III were significant lower than those in Gps II and IV at days 3 and 4, and at day 3 after transplantation, respectively (P < 0.05). The serum levels of IFN-γ, IL-2 and TNF-α were increased at day 21 to 28 after transplantation in all groups. The peak concentrations of IFN-γ, IL-2 and TNF-α in Gp III were significantly lower than those in Gps II and IV control groups at day 21 (P < 0.05).

CONCLUSIONCo-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD after allo-BMT in mice by inhibiting the early activation and expansion of donor T cells and reducing the serum levels of inflammatory cytokines.

Animals ; Bone Marrow Transplantation ; adverse effects ; Cytokines ; blood ; Female ; Forkhead Transcription Factors ; genetics ; Genetic Engineering ; Genetic Vectors ; Graft vs Host Disease ; immunology ; Lentivirus ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; cytology ; immunology ; Transduction, Genetic ; Transplantation, Homologous