Hamartoma ; Humans ; Laryngeal Diseases ; Male ; Middle Aged

OBJECTIVETo clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs).

METHODSTotal RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection.

RESULTSgdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells.

CONCLUSIONThis study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.

Animals ; Cloning, Molecular ; Gene Expression ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Male ; Mice ; Mice, Inbred Strains ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; metabolism ; Testis ; cytology ; metabolism ; Transfection

OBJECTIVETo investigate the role of endotoxin receptor expression in the activation of hepatic stellate cells (HSCs).

METHODSHSCs were isolated from normal rats and the expression of endotoxin receptors on quiet HSCs and in vitro activated HSCs was determined using RT-PCR and immunocytochemical staining methods. A rat model of liver fibrosis and cirrhosis was established. The expressions of CD14 and alpha-SMA in liver tissues were detected by immunohistochemical staining.

RESULTSFreshly isolated HSCs had a low level of CD14 mRNA expression and no expression of TLR4 mRNA was detected. The in vitro activated HSCs had increased expressions of CD14 mRNA and TLR4 mRNA and LPS up-regulated the expression of endotoxin receptors. Immunocytochemical staining showed cytoplasmic and nucleolus staining for CD14 in the cultured HSCs. LPS played a further role on CD14 protein expression. In the development of liver fibrosis, the number of CD14-positive cells in the livers was increased and these cells were distributed along the sinusoids. In the later stage of liver fibrosis, the CD14-positive cells were gathered in the fibrotic septae, which also contained alpha-SMA positive cells.

CONCLUSIONThe activated HSCs expressed endotoxin receptors. The endotoxin receptors may be involved in the role in which HSCs played in the inflammatory process and liver fibrosis development.

Actins ; metabolism ; Animals ; Cells, Cultured ; Hepatic Stellate Cells ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Liver Cirrhosis ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Receptors, Immunologic ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investegate the role of calcineurin (CaN) and its downstream nuclear factor of activated T-cells (NFATc3) in abdominal aorta restenosis following balloon dilatation in rats.

METHODSSD rats were randomly divided into sham-operated group, balloon group and cyclosporine A (CsA) group. The rats in the latter two groups were subjected to abdominal aorta injury with balloon dilatation, and those in CsA group were treated with CsA at the daily dose of 12.5 mg/kg from 3 days before the surgery to the end of the experiment. Thirty days afer the injury, histological analysis of the arterial wall was carried out with HE staining and immunohistochemistry. The expressions of CaN and NFATc3 in the abdominal aortas were detected with rea1-time PCR, and serum concentration of MCP-1 was determined using enzyme-linked immunosorbent assay.

RESULTSIntimal hyperplasia with irregular thickness of the neointima was observed in the aorta of rats with ballon injury. In rats with CsA treatment, the area of the intimal layers and the ratio of the intimal to the medial layers were obviously lower than those in the balloon injury group (P<0.05). Compared to those in the sham-operated group, the expressions of calcineurin protein and mRNA and NFATc3 mRNA in the arterial wall and serum level of MCP-1 increased significantly in the ballon injury group (P<0.05). CsA treatment significantly suppressed aorta restenosis and the alterations of CaN, NFATc3 and serum MCP-1 induced by ballon dilatation (P<0.05).

CONCLUSIONSCaN-NFATc3 signal transduction pathway mediates restenosis of rat abdominal aorta following ballon dilatation, and CsA can attenuate the restenosis by suppressing this pathway.

OBJECTIVETo investigate the effect of T-cell vaccination in murine experimental autoimmune hepatitis (EAH).

METHODSTo induce the EAH model, the syngeneic S-100 antigen emulsified in complete Freud's adjuvant was injected intraperitoneally to C57Bl/6 at day 1 and day 7. For T-cell vaccination, splenocytes were removed from animal 2 weeks after induction of EAH and from control animals, and activated in vitro by mitogen stimulation with Concanavalin A (Con A), then inactivated by mitomycin and injected at 5 10(7) cells per animal as T-cell vaccination at 14 and 7 days before first induction of EAH.

RESULTSThe histological grade and serum ALT level of the mice who received T-cell vaccination were decrease significantly, compared with that of model group (1.44+/-0.88 vs. 2.33+/-0.87, t=2.24, P<0.05; 63.0U/L+/-23.4U/L vs. 115.0U/L1+/-39.6U/L, t=2.37, P<0.01, respectively); there was no significant change in mice who received irrelevant T-cell vaccination.

CONCLUSIONT-cell vaccination with T cells from EAH animals, but not with irrelevant T cells, was able to protect animals from EAH.

Animals ; Hepatitis, Autoimmune ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes ; immunology ; Vaccination

The paper is aimed to investigate the effect of cyclosporine A (CyA) on the pharmacokinetics of ginkgolide B (GB) in rats, and to look for the mechanism of the changes in pharmacokinetic behaviors of GB. GB concentration in plasma, brain homogenate and urine samples of rats was determined by LC-MS. Effects of CyA on plasma levels, brain distributions as well as urinary excretions after intravenous administration of GB were evaluated. CyA co administrated intravenously at 10 mg kg(-1) or 20 mg kg(-1) significantly increased AUC(0-360 min) (P < 0.01) and decreased total CL of GB in rats. While co administrated CYP3A inhibitor itraconazole (ICZ) has no appreciable effect on the pharmacokinetic behavior of GB. CyA increased the brain uptake of GB in a dose-dependent manner. The brain distribution of GB was significantly increased at 5 min by different doses of CyA (P < 0.001), while at 20 and 60 min only high dose of CyA could significantly increase the levels of GB in the brain (P < 0.01 and P < 0.001). Different P-gp inhibitors CyA or verapamil (VER) or digoxin (DGX) decreased the urinary GB excretion, the urinary excretion of GB in 0-8 h were about 34.8% (P < 0.001), 59.4% (P < 0.001) and 79.7% (P < 0.05) of the control, separately. No appreciable effect of ICZ was observed on urinary excretion of GB. Coadministration of P-gp inhibitors CyA could significantly increase the plasma level, accelerate the brain distribution and decrease the urinary excretion of GB.
Animals ; Cyclosporine ; pharmacology ; Ginkgolides ; pharmacokinetics ; Herb-Drug Interactions ; Lactones ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tissue Distribution

BACKGROUNDOur previous studies demonstrated that mutant IkappaBalpha (IkappaBalphaM) inhibited the occurrence, growth and angiogenesis of human glioblastoma multiform (GBM). However, the specific mechanism by which IkappaBalphaM regulates protein-degrading enzymes secreted from GBM to inhibit invasion and metastasis has remained unclear. The aim of the present study was to investigate the regulatory role and significance of IkappaBalphaM genes in the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and matrix metalloproteinase (MMP)-9 in human GBM.

METHODSWe established the following four GBM cell lines stably expressing IkappaBalphaM by plasmid construction, gene transfection and screening for IkappaBalphaM protein expression: mutant IkappaBalpha-transfected cells (G36Delta-M), wild-type IkappaBalpha-transfected cells (G36Delta-W), empty plasmid transfected cells (G36Delta-P) and untransfected cells (G36Delta). The TIMP-2 and MMP-9 expression was detected by RT-PCR and Western blotting. Tumor cells were then implanted subcutaneously into nude mice to establish an animal model of ectopic tumor growth, and TIMP-2 and MMP-9 expression was determined by immunohistochemical methods.

RESULTSThe results showed that there was a significant increase in TIMP-2 expression and a significant decrease in MMP-9 expression in the G36Delta-M group at both the RNA and protein levels compared with the G36Delta-W group, G36Delta-P group and G36Delta group. Similar results were observed in the immunohistochemical staining analysis of tumor tissues. In the G36Delta-M group, TIMP-2 expression was significantly higher while MMP-9 expression was significantly lower than in the other three groups.

CONCLUSIONSOur findings indicate that IkappaBalphaM inhibits the activation of NF-kappaB. It significantly up-regulates TIMP-2 expression in human malignant glioma cells and down-regulates the expression of MMP-9. Thus, IkappaBalphaM maintains the integrity of the extracellular matrix and further inhibits the growth and metastasis of tumor tissues.

Animals ; Blotting, Western ; Cell Line, Tumor ; Glioblastoma ; genetics ; metabolism ; Humans ; I-kappa B Proteins ; genetics ; physiology ; Immunohistochemistry ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice ; Mice, Nude ; NF-KappaB Inhibitor alpha ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of tonifying Shen recipe (TSR) on levels of advanced glycosylation end products (AGEs) in aorta, serum lipids and lipid peroxidation in ovariectomized rats.

METHODSRats were randomly divided into the sham group, the ovariectomized group and the TSR group, in which the rats were treated with TSR for 13 weeks starting from 2 weeks after ovariectomy. Blood sample was taken out from rat at the end of the experiment after 24 hrs fasting for determination of lipids and lipid peroxidation, and the animal was sacrificed, the aorta was taken out for detecting AGEs.

RESULTSNo significant difference was found between groups in levels of total cholesterol and low density lipoprotein cholesterol. In comparing with the sham group, levels of aortic AGEs, serum triglyceride (TG), oxidized low density lipoprotein (OX-LDL) and malondialdehyde (MDA) in the ovariectomized group were obviously higher (P < 0.05 or P < 0.01), and levels of high density lipoprotein cholesterol (HDL-C), apo-lipoprotein A-I (apoA-I) and activity of superoxide dismutase (SOD) were lower (all P < 0.01). While in the TSR group, as compared with the ovariectomized group, the above-mentioned abnormal changes, excepting for TG, were all reversed to certain degree (P < 0.05 or P < 0.01).

CONCLUSIONTSR displays its cardiovascular protecting effect in ovariectomized rats through lowering the AGEs content in aorta, reducing the serum levels of OX-LDL and MDA, raising the levels of serum HDL-C and apoA-I and increasing SOD activity.

Animals ; Aorta ; metabolism ; Female ; Glycation End Products, Advanced ; metabolism ; Lipid Peroxidation ; drug effects ; Lipids ; blood ; Ovariectomy ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Adult ; Female ; Hamartoma ; complications ; diagnosis ; surgery ; Humans ; Kidney Calculi ; complications ; Spleen ; pathology ; surgery ; Splenectomy ; methods ; Splenic Diseases ; complications ; diagnosis ; surgery

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.
Animals ; Cryptorchidism ; metabolism ; Male ; Membrane Proteins ; analysis ; Mice ; Phosphatidylethanolamine Binding Protein ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; analysis ; Testis ; chemistry