Objective To stimulate the expansion of natural killer cells (NK) in vivo, to achieve high expression of Flt-3 ligand ( FL) in the mouse liver by hydrodynamic injection technology , to obtain high purity of NK for cell therapy and then to purify the spleen lymphocytes by MicroBeads sorting ( MACS ) technique.Methods C57BL/6 mice were repeatedly treated with hydrodynamic injection of FL expression plasmid .Then, surface markers of splenic NK were analyzed by flow cytometry and purified by MACS technique .Results Compared with mice treated with one hydrodynamic injection, the spleen of mice treated with two hydrodynamic injections showed significant variation, with the absolute num-ber of lymphocytes increased (6.02 ±1.15) times, the proportion of NK subsets increased (2.07 ±0.39) times, and the absolute number of NK subsets increased (12.49 ±2.39) times.Cell surface marker detection confirmed that NK activity and surface markers did not change significantly .NK with a purity of about (83.81 ±0.92)％ was obtained by the combination of two magnetic beads sorted with CD 5 ( Ly-1) MicroBeads Positive selection and EasySep TM Mouse NK Cell Isolation Kit negative selection .Conclusion NK can be expanded in the spleen of mice by hydrodynamic injection of FL plasmid without influence on the NK activity and surface markers.The combined use of CD5 (Ly-1) MicroBeads positive selection with EasySep TM Mouse NK Cell Isolation Kit negative selection can effectively remove T , B and other miscellaneous cells, thereby contributing to obtaining high-purity NK. This study provides good reference for the immunotherapy of tumor cells.

OBJECTIVETo investigate the changes of proto-oncogene c-fos, c-jun mRNA expression in angiotensin II (Ang II)-induced hypertrophy and effects of tanshinone II A (Tan) in the primary culture of neonatal rat cardiomyocytes.

METHODTwelve neonatal Wistar rats aged one day old of clean grade and both sexes were selected to isolate and culture cardiomyocytes. The cardiomyocytes were divided into: normal control group, Ang II (10(-6) mol x L(-1)) group, Ang II (10(-6) mol x L(-1)) +Tan (10(-8) g x L(-1)) group, Ang II (10(-6) mol x L(-1)) + valsartan (10(-6) mol x L(-1)) group, Tan (10(-8) g x L(-1)) group, valsartan (10(-6) mol x L(-1)) group. The cardiomyocyte size was determined by phase contrast microscope, the rate of protein synthesis in cardiomyocytes was measured by 3H-leucine incorporation. The c-fos, c-jun mRNA expression of cardiomyocytes were assessed using reverse transcription polymerase chain reaction (RT-PCR).

RESULTAng II was added to the culture medium and 30 min later, the c-fos, c-jun mRNA expression of cardiomyocytes increased significantly (P < 0. 01). After Ang II took effect for 24 h, the rate of protein synthesis in Ang II group increased more prominently than that in normal control group (P < 0.01). After Ang II took effect for 7 days, the size of cardiomyocyte in Ang II group increased obviously (P < 0. 05). If tanshinone II or valsartan was added to the culture medium before Ang II, both of them could inhibit the increase of c-fos, c-jun mRNA expression (P < 0.01), cardiomyocyte protein synthesis rate (P < 0.01), and cardiomyocyte size (P < 0.05) induced by Ang II.

CONCLUSIONTanshinone II could ameliorate Ang II-induced cardiomyocytes hypertrophy by inhabiting c-fos, c-jun mRNA expression.

Angiotensin II ; biosynthesis ; pharmacology ; Animals ; Cardiomegaly ; chemically induced ; metabolism ; pathology ; Diterpenes, Abietane ; Gene Expression Regulation ; drug effects ; Genes, fos ; genetics ; Genes, jun ; genetics ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Phenanthrenes ; pharmacology ; Proto-Oncogene Proteins c-fos ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan

BACKGROUNDThere are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains.

METHODSThe right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation.

RESULTSGrafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded.

CONCLUSIONCultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain.

Animals ; Astrocytes ; transplantation ; Brain ; pathology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cerebral Infarction ; metabolism ; pathology ; therapy ; Fetal Tissue Transplantation ; Glial Fibrillary Acidic Protein ; analysis ; Humans ; Microtubule-Associated Proteins ; analysis ; Neocortex ; cytology ; Neurons ; transplantation ; Rats ; Synaptophysin ; analysis

AIMTo assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.

METHODSThe total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.

RESULTSThe pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1.

CONCLUSIONhHSF1 can transcriptionally regulate prohibitin expression.

Base Sequence ; DNA-Binding Proteins ; physiology ; Gene Expression Regulation ; HEK293 Cells ; Heat Shock Transcription Factors ; Humans ; Molecular Sequence Data ; Promoter Regions, Genetic ; Repressor Proteins ; genetics ; Transcription Factors ; physiology ; Transcription, Genetic ; Transfection

OBJECTIVETo observe the basic characteristics of rats with subluxation, which was hypomobility of motion.

METHODSOne hundred and eight male SD rats (weighted, 350 to 450 g) were randomly divided into experimental group (simple fixation and rotatory fixation) and sham operation group. Each group was subdivided into four groups, including 2, 4, 8 and 12 weeks sub-groups. Simple fixation and rotatory fixation group were modeled by implanting external linked fixation system; Sham operation group was dealed with operation intervention. At the end of connection, X-ray films was used to observe posterier body angle (PBA); Spinal stiffness system for stiffness in simple fixation group and rotatory fixation group.

RESULTSRadiographic results showed that variation of PBA n experimental groups tended to decrease; and there was no significant difference between simple fixation group and rotatory fixation group at the end of linking time (P > 0.05). But there was significant difference between the experimental group and sham operation group (P < 0.05). The degree of stiffness showed that the stiffness in experimental group increased with the linking time, but no meaning between groups (P > 0.05).

CONCLUSIONRats with subluxation induced by external linked fixation system can effectively change biomechanical characteristics of spine, reduced the motion with linking time.

Animals ; External Fixators ; Fracture Fixation ; Humans ; Joint Dislocations ; physiopathology ; surgery ; Lumbosacral Region ; injuries ; physiopathology ; surgery ; Male ; Range of Motion, Articular ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo review the outcome of repeated percutaneous sperm aspiration (PESA) and testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI).

METHODSForty-three cycles of 31 cases of azoospermic patients which underwent at least two PESA or TESE for ICSI from January 2001 to December 2002 were collected. The sperm retrieval, fertilization, implantation and clinical pregnancy were analyzed.

RESULTSTwenty-four cases underwent PESA and 7 cases underwent TESE. There were not any complications in these patients. Compared with the first cycle of 154 cases, the fertilization rate, implantation rate and clinical pregnancy rate were 78.39% vs 73.64%, 19.68% vs 18.38% and 34.88% vs 37.91%, respectively(P > 0.05).

CONCLUSIONSRepeated PESA or TESE is safe and well tolerated in azoospermic patients. Compared with the first cycle, the differences of repeated PESA or TESE cycles in fertilization rate, implantation rate and clinical pregnancy rate were not statistically significant.

Adult ; Azoospermia ; therapy ; Female ; Humans ; Male ; Middle Aged ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate ; Sperm Injections, Intracytoplasmic ; methods ; Tissue and Organ Harvesting ; methods

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

OBJECTIVETo establish a pig model of chronic external counterpulsation.

METHODSTwelve pigs were anaesthetized with sodium pentobarbital (< or =30 mg/kg.b.w.) and 846 mixture (< or =0.1 ml/kg.b.w.) and counterpulsed in a lateral position for 2 h every two days (totally 36 h) with 0.025 to 0.04 MPa/cm(2) pressure.

RESULTSExternal counterpulsation was successfully completed in all the animals. Combined administration of sodium pentobarbital and 846 mixture resulted in good anesthetic effect with reduced anesthetic dosage and minimal side effect on the viscera (the liver, kidney and heart, etc).

CONCLUSIONThe pig model of chronic external counterpulsation has been successfully established. Combined use of sodium pentobarbital and 846 mixture is recommended for chronic external counterpulsation.

Anesthesia, General ; methods ; Animals ; Animals, Newborn ; Assisted Circulation ; Counterpulsation ; methods ; Models, Animal ; Pentobarbital ; administration & dosage ; Swine

OBJECTIVESTo review the retrospective treatment results of the azoospermia patients during January 2001 to January 2002 in the fertility center.

METHODSOne hundred males attempted intracytoplasmic sperm injection (ICSI) cycle for treatment of azoospermia. All patients were undergone sperm retrieval by percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated.

RESULTSSperm were retrieved by PESA in 76 of 100 (76%) and by TESE in 23 of 100 (23%) men of azoospermia. The fertilization rate, implantation rate and clinical pregnancy rate were 71.3%, 20.35% and 42.11% respectively in PESA group, and 75.18%, 22.05% and 41.60% respectively in TESA group. Thirty-two clinical pregnancies were achieved with 15 ongoing pregnancies and subsequent live delivery for 15 cases in PESA group, and 2 cases of miscarriage, while 10 clinical pregnancies were achieved with 6 ongoing pregnancies, 2 cases of live delivery and 2 cases of miscarriage in TESA group. One case failed to retrieve sperm by TESE and canceled.

CONCLUSIONSHormonal levels and testicular histology are unable to predict which men with azoospermia will have sperm retrieved by PESA and TESE. PESA and TESE with ICSI are effective methods to treat azoospermia. There were no significant differences in fertilization, implantation and clinical pregnancy rate between two groups.

Adult ; Female ; Follicle Stimulating Hormone ; blood ; Humans ; Luteinizing Hormone ; blood ; Male ; Middle Aged ; Oligospermia ; blood ; therapy ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic ; methods

We report the clinical characteristics, treatments and outcomes of 4 rare cases of mixed amanita fuliginea and amanita rimosa poisoning with rhabdomyolysis, and review the research progress in the intoxication mechanism and treatment. The latent time of amanita poisoning, defined as the period from the ingestion of poisonous mushroom to the onset of gastrointestinal symptoms, was about 8 days, and the severity of poisoning was associated with the amount of mushroom ingested. All the 4 patients developed multiple organ dysfunctions within 3 to 4 days after mushroom ingestion, predominantly in the liver, kidney and central nervous system accompanied with acute gastrointestinal injury and rhabdomyolysis. The treatment measures included persistent hemofiltration and intermittent hemoperfusion once daily for 5-7 days, and plasma exchange was administered in 2 cases for 1 or 2 times. High-dose vitamin C, glucose and corticosteroid were also given to the patients. After the treatments, two patients were cured and the other two died due to an excess intake of poisonous mushroom and lack of early preemptive therapies. Early emetic, gastric lavage, catharsis, fluid infusion and diuresis are critical to interrupt the enterohepatic circulation of amanita phalloides toxins and prevent the development of multiple organ dysfunction. Enhanced hemofiltration and sequential plasma therapy might effectively eliminate toxin from the blood to protect against further organ damages.