OBJECTIVETo investigate the effect of small hairpin interfering RNA (shRNA) in suppressing cytochrome P450 3A4 (CYP3A4) gene expression in CHL-3A4 cells.

METHODSThree shRNA expression vectors targeting CYP3A4 gene (CYP3A4 I, C YP3A4 II, and CYP3A4 III, respectively) were designed, synthesized and transfected into CHL-3A4 cells via liposomes. The inhibitory effect of shRNA on CYP 3A4 gene expression was detected by Western blotting and RT-PCR, and the effect of shRNA transfection in suppressing cyclophosphamide-induced cytotoxicity was measured using MTT assay.

RESULTSThe vector carrying CYP3A4 III shRNA significantly reduced the expression of CYP3A4 gene at both the mRNA (75%) and protein levels (80%) in CHL3A4 cells. The cytotoxicity of cyclophosphamide was markedly inhibited by CYP3A4 III-mediated suppression of CYP3A4 gene expression by 75% in CHL-3A4 cells.

CONCLUSIONThe vector-mediated RNA interference can suppress CYP3A4 gene expression in CHL-3A4 cells, and RNA interference technique provides a new means for studying cytochrome P450 gene function in mammalian cells.

Animals ; Cells, Cultured ; Cricetinae ; Cytochrome P-450 CYP3A ; genetics ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung ; cytology ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo evaluate the associations of human leukocyte antigen (HLA) DQB1 gene with onset age and autoantibodies in type 1 diabetes mellitus(T1DM) in Chinese Han population in Sichuan area.

METHODSForty-six type 1 diabetic patients and 52 healthy control subjects were involved in this study. HLA-DQB1 typing was performed by polymerase chain reaction-sequence specific primer(PCR-SSP). Glutamic acid decarboxylase antibody (GADA) and islet cell antibody (ICA) were qualitatively analyzed by enzyme linked immunosorbent assay (ELISA).

RESULTSThe positive rate of DQB1*0201 was higher in T1DM than in controls (OR=18, P<0.005), but those of DQB1*0601, *0602 were higher in controls than in T1DM(OR=0.07, 0.31 respectively, both P<0.05).The positive rate of DQB1*0602 in type 1 diabetic patients with onset age>or=20 years was higher than that in the patients with onset age <20 years (P<0.05). GADA was more frequent in DQB1*0201(+) patients than in DQB1*0201 (-) patients (P<0.025).

CONCLUSIONThe findings show that DQB1*0201 is susceptible to T1DM, whereas DQB1*0601, *0602 are protective in Chinese Han population in Sichuan area. DQB1*0602 may delay the onset of T1DM. The positive rate of DQB1*0201 correlates positively with that of GADA.

Adolescent ; Adult ; Age of Onset ; Autoantibodies ; immunology ; Child ; Child, Preschool ; China ; epidemiology ; Diabetes Mellitus, Type 1 ; epidemiology ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Predisposition to Disease ; genetics ; Glutamate Decarboxylase ; immunology ; HLA-DQ Antigens ; genetics ; HLA-DQ beta-Chains ; Humans ; Male ; Membrane Glycoproteins ; genetics ; Middle Aged ; Polymerase Chain Reaction ; Young Adult

OBJECTIVETo observe the base of the interference in correlated biotic active matter obtained multi-drug resistance induced by chemotherapy for different alkaloid, and to supervise the use in clinic to restrain the multi-drug resistant of chemotherapy, and thereby to improve the curative effect.

METHODAfter bestowing subter-dosage unite chemotherapeutant to ascites S180 mouse to set up the mouse models of multi-drug resistance of S180 tumour cell, and giving the mouse matrine, terandrine, oxymatrine and berberine hydrooh loride for 4 weeks, the P170, LRP, TOPOII, Fas and apoposis were determined by flow cytometry.

RESULTMatrine and terandrine could obviously reduce the express of P170, LRP and the activation of TOPOII, and increase the ratio of the express of Fas and the apoposis of drug resistant tumour cell. And at the same time it could obviously reduce the express of intercellular adhesion molecule(CD54).

CONCLUSIONMatrine and terandrine can interfere in MDR which results from chemotherapeutics by the adjustment of correlated biotic active matter, besides, the different degree of alkaloid effect with different configuration.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Alkaloids ; isolation & purification ; pharmacology ; Animals ; Apoptosis ; drug effects ; Benzylisoquinolines ; isolation & purification ; pharmacology ; Berberine Alkaloids ; isolation & purification ; pharmacology ; DNA Topoisomerases, Type II ; metabolism ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Female ; Male ; Mice ; Plants, Medicinal ; chemistry ; Quinolizines ; isolation & purification ; pharmacology ; Random Allocation ; Sarcoma 180 ; metabolism ; pathology ; Tumor Cells, Cultured ; Vault Ribonucleoprotein Particles ; metabolism ; fas Receptor ; metabolism

Objective To analyze the clinical value of minimal residual disease(MRD) of acute myeloblastic leukemia(AML) with AML 1-ETO by using the real-time quantitative reverse transcriptase polymerase chain reaction(RQ-RT-PCR).Methods From Jan.2001 to Jan.2007,the MRD of 32 AML1-ETO-positive AML patients were analyzed by using PQ-RT-PCR.The detection of the AML1-ETO was taken after the induced chemotherapy every 1.5-2.0 months during the consolidation therapy.The survival of different stages in children with AML was analyzed by SPSS 10.0 software and calculated by using Kaplan-Meier analysis.Results Thirty-two patients received the induced chemotherapy and 29 patients with complete remission morphologically,3 patients had no complete remission morphologically and then gave up.Patients with molecular remission were associated with a high probability of survival(P=0.001 8).Patients with high transcript levels at diagnosis had no difference in event free survival with patients with low transcript levels.The quality of molecular response after induction,6 months in the chemotherapy as well as consolidation period,has significant impact on the event free survival(P=0.023,0.000 1,0.004 9).Conclusion The current study demonstrate that quantitative evaluation of AML1-ETO transcript levels is important and may be helpful for therapeutic decisions in future.

Objective To summarize the experience of long-term survival in patients after simulta- neous kidney-pancreas transplantation(SKPT)with modified enteric drainage(ED).Methods From October 2001 to July 2004,6 patients with end-stage renal disease due to Type 1 diabetes underwent SKPT with modified ED,ie,side-to-side anastomosis between the duodenum of donors and jejunum of recipients. The medication regimen included:mycophenolic acid 500 mg and tacrolimus 2 mg before operation;methyl- prednisolone(MP)1.0 during operation;and 2-dose anti-IL-2 receptor monoclonal antibody(2 cases)or antihuman thymocyte globulin(ATG)(4 cases)for immune induction therapy;MP was used on the first 3 d after transplantation,triple immunosuppressive therapy(tacrotimus,mycophenolic acid and prednisone)was used on the second d after transplantation.Anticoagulants such as low molecular heparin or alprostadil were used for 7-10 d to prevent thrombosis in pancreas graft.Somatostatin was used as prophylaxis for graft pan- creatitis.Ganciclovir was used to prevent cytomegalovirus infection when renal graft gradually recovered 3 to 5 d after transplantation.The follow-up was from 1 year and 3 months to 4 years and 1 month.Results Transplantation was successful in all 6 cases.The blood sugar levels were 6-16 mmol/L.Low-dose insulin was used for 5-10 d,then the blood sugar levels returned to normal range.One of 6 patients experienced nephrotoxicity because of high tacrolimus blood concentration at 7 d after operation;after 3 dialyses and re- duction of tacrolimus dose,the renal allograft regained normal function.Three cases experienced alimentary tract hemorrhage at 14,20 and 22 d,respectively,after operation;the bleeding was stopped after treatment. There were no complications such as pancreatic fistula,intestinal fistula and thrombosis early after operation. All the patients are now alive,specifically,1 survived over 4 years,3 over 3 years,1 over 2 years,and 1 over 1 year.All had normal blood sugar free of insulin use.Five cases had normal renal graft function,with normal sCr,and 1 had sCr＞400?mol/L. Two cases were admitted to hospital due to upper respiratory infection and furuncles in the skin of head 6 months and 2 years,respectively,after operation.They were both cured.No complications such as urinary infection,metabolic acidosis and dehydration occurred.Conclusions SKPT is effective for the treatment of end-stage renal disease due to Type 1 diabetes.SKPT with modified ED are relatively simple with physiological compatibility and fewer complications.High quality of donated organs, HLA matching,pancreatic drainage pattern,rational periopcrative medications and infection late after trans- plantation are important factors affecting the long-term survival of the patients.

OBJECTIVEHemolytic uremic syndrome (HUS) is a common primary disease that can cause acute renal failure in childhood. Renal disease is the most important long-term complication in patients who survived the acute stage of HUS. Use of angiotensin-converting enzyme inhibitors (ACEI) and a restricted protein intake may be beneficial to the patients. However, it is not established whether such patients should be treated with steroids and immunosuppressors. The present study aimed to probe into the benefit of using steroid and immunosuppressor in patients after acute stage of HUS.

METHODSThe subjects included 17 patients (aged 9 months to 15 years, 12 males, 5 females) with HUS. Thirteen patients recovered from the acute stage of HUS, and underwent continuative treatment and follow-up. All the patients were treated with ACEI and early restriction of protein intake. Additionally, 2 children manifested as glomerulonephritis, one was treated with triperygium glycosides. Other 11 children who manifested as nephrotic syndrome were treated with prednisone, among them 5 children had no response or had incomplete response to prednisone, for these children short-term high dose cyclophosphamide or methylprednisolone pulse treatment were added; in 3 of the children short-term high dose methylprednisolone treatment was applied additionally for membranoproliferative glomerulonephritis and/or focal segmental glomerulosclerosis and crescentic glomerulonephritis.

RESULTSAfter follow-up for 2 months to 8 years, 4 patients with milder disease recovered, their blood pressure, renal function and urinalysis became normal, but 1 patient had recurrence. Among 9 patients with severe disease, 6 maintained normal blood pressure, recovered renal function and urinalysis, the other 3 patients failed to comply with treatment protocol and died during the 3rd, 9th and 13th month. The remainder (4 cases) gave up therapy and died on the 27th to 48th days of the course.

CONCLUSIONThe treatment applied in this study could improve the prognosis of patients after acute phase of HUS evidently by using the steroid and immuno suppressor according to clinical classification and pathological findings. It is recommended that triperygium glycosides is beneficial to children with glomerulonephritis, proteinuria and hematuria after acute stage of HUS. Adjustment of therapeutic schedule based on pathological findings after renal biopsy is helpful. To the patients with progressive renal failure who have no response to the steroid and immunosuppressors, steroid and immunosuppressor should be discontinued and dialysis treatment should be applied. Protocol compliance is also an important factor.

Acute Disease ; Adolescent ; Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Child ; Child, Preschool ; Combined Modality Therapy ; Diet, Protein-Restricted ; Drug Therapy, Combination ; Female ; Follow-Up Studies ; Hemolytic-Uremic Syndrome ; diet therapy ; drug therapy ; physiopathology ; Humans ; Immunosuppressive Agents ; therapeutic use ; Infant ; Male ; Prognosis ; Steroids ; therapeutic use ; Treatment Outcome

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Leukemic ; drug effects ; Genes, Regulator ; drug effects ; Humans ; Interferon Regulatory Factor-1 ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; STAT2 Transcription Factor ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo compare the long-term effect and safety of tacrolimus (FK506) and cyclosporine (CsA) in kidney transplant (KT) recipients carrying hepatitis B Virus(HBV).

METHODSA total of 109 patients with HBV were randomized into FK506 group (52 cases) and CsA group (57 cases) after KT, and a 2-year-long follow-up of the patients was conducted to record the patient and graft survival, incidence of acute graft rejection and postoperative liver function.

RESULTSThe 2-year patient/graft survival was 86.0%/73.7% and 94.2%/90.3% in CsA and FK506 groups, respectively (P<0.05), with incidence of acute rejection of 10.5% and 9.6% (P>0.05), and rate of abnormal liver function of 26.3% and 15.4% (P<0.05), respectively. Eight patients (14.4%) in CsA group required a drug conversion but none in FK506 group. The drug conversion resulted in significant reduction of ALT/AST level from 255.13+/-31.38/201.88+/-21.25 U/L to 31.25+/-11.50/25.13+/-9.68 U/L (P<0.01).

CONCLUSIONFor HBV-carrying renal transplant recipients, FK506 as the primary choice of immunosuppressant can be more effective and safer than CsA.

Adolescent ; Adult ; Carrier State ; physiopathology ; Cyclosporine ; administration & dosage ; adverse effects ; pharmacology ; Drug-Related Side Effects and Adverse Reactions ; Female ; Graft Rejection ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; Humans ; Kidney Transplantation ; adverse effects ; Liver ; drug effects ; physiology ; Male ; Middle Aged ; Tacrolimus ; administration & dosage ; adverse effects ; pharmacology ; Young Adult

In order to investigate the effect of shRNA targeted to beta-catenin on the growth of K562 cells, plasmid containing beta-catenin specific shRNA sequence was transfected into K562 cells by lipofectamine 2000, and G418 was added to screen the positive cells. Real-time PCR and Western blot were used to detect the expression of beta-catenin. Cell growth curve, MTT and colony forming cell assays were used to evaluate the proliferation potential of cells. The results showed that the mRNA level of beta-catenin was reduced significantly in K562 cells transfected into interfering plasmid as compared with control plasmid, while the protein level failed to demonstrate difference by the time of 72 hours after transfection. After long-term culture with G418, the count of positive cells enhanced in control group while no positive cells survived in the interfering group. Colony-forming cell assays revealed that the K562 cells in interfering group formed colonies with very small size and low forming rate, compared with the control group, though the growth curve and MTT failed to illustrate differences. It is concluded that the beta-catenin-specific shRNA mediated by plasmid can effectively knockdown the expression of beta-catenin gene and inhibit the colony-forming ability in K562 cells, it is a potential target for the therapy of CML, even in blast crisis.
Cell Proliferation ; Humans ; K562 Cells ; Neoplastic Stem Cells ; Plasmids ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; beta Catenin ; genetics ; metabolism

OBJECTIVETo explore the categories of drugs causing hepatotoxicity and analyze the clinical and histological features of the corresponding drug-induced liver injury (DILI), in order to gain insights into potential diagnostic factors for DILI.

METHODSA total of 138 DILI patients treated at our hospital from April 2008 to April 2010 were retrospectively analyzed. The responsible drug for each DILI case was recorded. The Roussel Uclaf Causality Assessment Method (RUCAM) had been used to diagnose DILI. Only cases that had scored as highly probable or probable (more than or equal to 6 points by RUCAM) were included in this study. The patients' general condition, clinical manifestations, and serum biochemical and immunological parameters were assessed. Sixty-six of the patients underwent liver biopsy, and were assessed for liver pathological changes. Clinical and laboratory test data were collected and used to classify the total 138 cases as hepatocellular injury, cholestatic, or mixed hepatocellular-cholestatic types.

RESULTSWithin our patient population, the leading cause of DILI was Chinese herb medicine, accounting for 53.62% of cases. Antibiotics were implicated in 7.97% of cases, and dietary supplement in 6.52% of cases. Correlation between the clinical features and histological injury pattern was stronger at the time of biopsy (more than or equal to 3 days after laboratory results) (kappa = 0.63, P less than 0.05) than at the onset of DILI (kappa = 0.25, P less than 0.05). All modified hepatic activity index (HAI) necroinflammatory scores and fibrosis scores were more severe in the cholestatic and mixed injury types than in the hepatocellular injury type (P less than 0.01 and P less than 0.05, respectively).

CONCLUSIONChinese herbal medicine, dietary supplements and antibiotics were the main causes of DILI in our patient population. The clinical and histological features correlated well, especially at later stages of DILI. The degree of inflammation and fibrosis was significantly higher in cholestatic and mixed hepatocellular-cholestatic injury types than in the hepatocellular injury type. Assessment of both clinical and pathological features may represent a more accurate diagnostic method for DILI.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anti-Bacterial Agents ; adverse effects ; Anti-Infective Agents ; adverse effects ; Chemical and Drug Induced Liver Injury ; pathology ; Drugs, Chinese Herbal ; adverse effects ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Young Adult