OBJECTIVETo study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).

METHODSCbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.

RESULTSCbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.

CONCLUSIONSNew Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.

Alkaline Phosphatase ; biosynthesis ; genetics ; Animals ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; biosynthesis ; genetics ; Fibroblasts ; cytology ; physiology ; Gene Expression ; Genetic Vectors ; Mice ; Osteocalcin ; biosynthesis ; genetics ; Osteogenesis ; genetics ; physiology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo evaluate the outcome of emergency adult right lobe living donor liver transplantation for fulminant hepatitis.

METHODSNine cases of adult right lobe living donor liver transplantation were performed from September 2002 to August 2005, the clinical and follow-up data was analyzed.

RESULTSAccording to Child Pugh Turcotte (CPT) classification, 9 patients were classified as grade C before transplant. The Model for End-Stage Liver Disease (MELD) scores of these patients were 26.7 +/- 8.8. The principal pre-transplant complications included hepatic encephalopathy (5 cases), electrolyte disturbance (3 cases), renal failure (2 cases), gastrointestinal bleeding (1 case). The operations in donors and recipients were all successful. The post-transplant complications induced pulmonary infection in 2 patients, acute renal failure in 3 and transplantation related encephalopathy in 1. There were no primary graft non-function and no blood vessel and bile tract complications occurred. One-year survival rate was 55.6%. No serious complication or death found in donors.

CONCLUSIONSEmergency adult to adult living donor liver transplantation is an effective treatment for fulminant hepatitis but the safety of the donors should be assessed strictly preoperation.

Adult ; Critical Illness ; Emergency Medical Services ; Female ; Follow-Up Studies ; Hepatitis ; pathology ; surgery ; Humans ; Liver Transplantation ; methods ; Living Donors ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharmacokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C(max0, AUCO(0-t), AUC(0-∞) were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 μmol x L(-1) Danshensu. The IC50 parameters was (53.04 ± 2.43) μmol x L(-1). The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvastatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94)% and (63.61 ± 3.94)%, respectively, by Danshensu at 1 and 10 μmol x L(-1). While transport of rosuvastatin was reduced by (8.22 ± 2.40)% and (11.56 ± 3.04)% and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATPJBI. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence.
Animals ; Drug Interactions ; Drugs, Chinese Herbal ; pharmacology ; HEK293 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lactates ; pharmacology ; Organic Anion Transporters ; metabolism ; Rats ; Rosuvastatin Calcium ; pharmacology ; Solute Carrier Organic Anion Transporter Family Member 1b1

BACKGROUNDbeta-glucan is the major structure component of Candida albicans (C. albicans) cell wall. It has been demonstrated that Dectin-1 as the principal C-type lectin pattern-recognition receptor (PRR) can recognize fungal beta-glucan and induce immune responses. In this study, we sought to clarify whether insoluble beta-glucan from the cell wall of C. albicans (CaIG) could induce immune responses in human THP-1 monocytes (a human acute monocytic leukemia cell line) and to determine the underlying mechanisms.

METHODSHuman THP-1 monocytes were challenged with CaIG in vitro. The mRNA expression of Dectin-1, Toll-like receptors (TLR2), proinflammatory cytokine (TNF-alpha) and chemokine (IL-8) was assayed by real-time reverse transcription polymerase chain reaction (RT-PCR). The secretion of TNF-a and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA). H(2)O(2) release was determined by microplate fluorescent assay. Western blotting was used to analyze IkappaB-a phosphorylation and degradation.

RESULTSExposure of THP-1 monocytes to CaIG led to increased gene expression and secretion of TNF-alpha and IL-8. CaIG induced H(2)O(2) release in a time-dependent manner. CaIG hydrolyzed with zymolyase failed to induce gene expression and secretion of TNF-alpha, IL-8 and H(2)O(2) release. CaIG up-regulated the mRNA of Dectin-1, whereas the mRNA level of TLR2 was not altered. THP-1 monocytes challenged with CaIG resulted in the activation of NF-kappaB in a time-dependent manner. Dectin-1 inhibitor laminarin blocked the CaIG-induced production of TNF-alpha and H(2)O(2) in THP-1 monocytes, but no such effect was observed in pretreatment with anti-TLR2 neutralizing antibody and the LPS inhibitor (polymyxin B).

CONCLUSIONCaIG may play a role in activation of immune responses in human THP-1 cells through Dectin-1, not TLR2.

Blotting, Western ; Candida albicans ; metabolism ; Cell Line, Tumor ; Cell Wall ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Humans ; Hydrogen Peroxide ; metabolism ; Interleukin-8 ; genetics ; metabolism ; Lectins, C-Type ; Membrane Proteins ; genetics ; metabolism ; Monocytes ; drug effects ; immunology ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; genetics ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; beta-Glucans ; pharmacology

OBJECTIVETo detect sperm mitochondrial membrane potential (MMP) of varicocele patients and investigate its clinical significance.

METHODSSixty-seven varicocele patients were divided into a VC1 (grade 1, n = 26), a VC2 (grade 2, n = 21) and a VC3 group (grade 3, n = 20). And 29 normal fertile volunteers were included in a control group ( m = 29). Conventional semen analyses were performed by computer-assisted semen analysis (CASA). Semen samples were washed, followed by JC-1 staining to evaluate the sperm MMP (JC-1+ %) by flow cytometry.

RESULTSThe sperm MMPs of the VC1, VC2 and VC3 groups were siginificantly lower ([56.29 +/- 16.32]%, P < 0.05; [45.04 +/- 13.21]%, P < 0.01; [31.63 +/- 12.91]%, P < 0.01) than that of the control ([76.21 +/- 13. 96]%). There was a significant positive correlation between the percentage of JC-1+ and that of grade (a + b) sperm (r =0.693, P=0.000).

CONCLUSIONThe decreased MMP in the sperm of varicocele men might be one of the important causes of male infertility.

Adult ; Flow Cytometry ; Humans ; Male ; Membrane Potential, Mitochondrial ; Sperm Motility ; Spermatozoa ; physiology ; Varicocele ; metabolism ; physiopathology ; Young Adult

OBJECTIVETo study the pathologic features of dilated heart in cardiac transplant recipients, with clinicoradiologic correlation.

METHODSSixty recipient hearts from cardiac transplantation performed in Fuwai Hospital were analyzed by gross examination, histologic observation and electron microscopy. Clinicoradiologic correlation was available in 40 cases.

RESULTSAmongst the 40 cases of dilated heart, 52.5% (21/40) were due to dilated cardiomyopathy, 22.5% (9/40) due to arrhythmogenic right ventricular cardiomyopathy, 15.0% (6/40) due to ischemic cardiomyopathy, and the remaining 10.0% (4/40) due to miscellaneous causes, including local noncompaction of ventricular myocardium, giant cell myocarditis, alcoholic cardiomyopathy and hypertensive cardiomyopathy. The discrepancy rate between clinical and pathologic diagnosis was 37.5% (15/40). The erroneous categories included arrhythmogenic right ventricular cardiomyopathy (7 cases), ischemic cardiomyopathy (5 cases), and giant cell myocarditis (1 case), which were all mistaken clinically as dilated cardiomyopathy. While ischemic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, noncompaction of ventricular myocardium and giant cell myocarditis have distinctive pathologic features, the diagnosis of alcoholic and hypertensive cardiomyopathies required clinicopathologic correlation. Dilated cardiomyopathy due to viral myocarditis was not identified in the cases studied.

CONCLUSIONPathologic examination is essential in analysis of transplant recipient heart and helps to rectify clinical diagnostic discrepancy.

Adolescent ; Adult ; Arrhythmogenic Right Ventricular Dysplasia ; diagnosis ; pathology ; Cardiomyopathy, Alcoholic ; diagnosis ; pathology ; Cardiomyopathy, Dilated ; diagnosis ; pathology ; Diagnostic Errors ; Dilatation, Pathologic ; diagnosis ; pathology ; Female ; Giant Cells ; pathology ; Heart Transplantation ; pathology ; Humans ; Hypertension ; complications ; Male ; Middle Aged ; Myocardial Ischemia ; diagnosis ; pathology ; Myocardium ; pathology

OBJECTIVETo investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.

METHODSTransfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.

RESULTSIn group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.

CONCLUSIONTrue bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

Animals ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; Disease Models, Animal ; Fibroblasts ; cytology ; metabolism ; Plasmids ; genetics ; Rabbits ; Radius ; injuries ; physiopathology ; surgery ; Random Allocation ; Skin ; cytology ; Tissue Engineering ; methods ; Transfection

The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharma­cokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*1a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C_max, AUC_0-t, AUC_0-∞ were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL_z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 μmol·L^-1 Danshensu. The IC₅₀ parameters was (53.04 ± 2.43) μmol·L^-1. The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvas­tatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94) % and (63.61 ± 3.94) %, respectively, by Danshensu at 1 and 10 μmol·L^-1. While transport of rosuvastatin was reduced by (8.22 ± 2.40) % and (11.56 ± 3.04) % and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATP1B1. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence.