Objective To evaluate the role of AMP-activated protein kinase (AMPK)-dependent autophagic signaling pathway in ketamine-induced reduction of diabetic neuropathic pain (DNP) in rats.Methods Sixty male Wistar rats,aged 3 months,weighing 200-250 g,were equally randomized into 5 groups using a random number table:control group (C group),normal saline group (NS group),ketamine group (K group),ketamine + Compound C group (KC group),and ketamine + 3-methyladenine (3-MA) group (KM group).DNP model was established by intraperitoneal injection of streptozocin (STZ)65 mg/kg in anesthetized rats.Four weeks later,the equal volume of normal saline,ketamine 10 mg/kg,ketamine 10 mg/kg + Compound C1 mg/kg,and ketamine + 3-MA 2 μl were injected intraperitoneally once a day for 7 consecutive days in NS,K,KC and KM groups,respectively.The mechanical paw withdrawal threshold (MWT) was measured on 8th day.The rats were then sacrificed,and the lunbar segment (L1-5) of the spinal cord was removed for determination of the expression of AMPKαt,Beclin-1,microtubule-associated protein 1 light chain (LC) 3B (by Western blot) and dendritic spine density in the dorsal root ganglia.Results Compared with group C,the MWT,expression of AMPKα,Beclin-1,and LC3B,and dendritic spine density were significantly decreased in group NS (P＜0.05).Compared with group NS,the MWT,expression of AMPKαt,Beclin-1,and LC3B,and dendritic spine density were significantly increased in group K (P＜0.05).Compared with group K,the MWT,expression of AMPKα,Beclin-1,and LC3B,and dendritic spine density were significantly decreased in KC and KM groups (P＜0.05).Conclusion Activation of AMPK-dependent autophagic pathway is involved in ketamine-induced reduction of DNP in rats.

Objective To explore the feasibility of exogenous NGF and/or Noggin gene transfecting into the bone marrow-derived mesenchymal stem cells (BMSCs) and observe the differentiation of BMSCs modified by NGF and/or Noggin.Methods BMSCs were isolated from SD rat and purified by adherent method and these cells were identified by their phenotypical properties and their abilities of differentiating into adipocytes.Ad-GFP-NGF and/or Ad-GFP-Noggin were transfected into BMSCs.The protein expressions of NGF and/or Noggin were detected using immunocytochemistry and Western blotting.The differentiations of gene modified BMSCs were observed by immunohistochemistry.Result The cells selected by adherent method had basic phenotypical properties of BMSCs and could differentiate into adipocytes.BMSCs without transfection and those transfected with Ad-GFP expressed low level of NGF without Noggin expression.All gene modified BMSCs could express NGF and/or Noggin.After transfection, BMSCs could differentiate into cells having neuronal morphology and expressing NF (H).The combined transfection group had the highest ratio of NF(H)+ cells among all the groups.Conclusion BMSCs can be isolated and purified from rat bone marrow by adherent method.Ad-GFP-NGF and/or Ad-GFP-Noggin can transfect BMSCs safely and the transfected cells can express those proteins persistently and efficiently.NGF and Noggin can induce BMSCs into neuron-like cells in vitro and when they exist simultaneously,the differentiation is further enhanced.

OBJECTIVEWe investigated the in vivo effects of recombinant adenovirus-associated virus type-2 (AAV-2) mediated interleukin-10 (IL-10) gene transfer on the expression of matrix metalloproteinase (MMP)-2, 9, tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I and type III in a rat acute myocardial infarction model.

METHODMale Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 6): sham operation group, MI/AAV2 group, and MI/AAV2-IL-10 group (10(10) vg/ml x 0.1 ml injection at peri-infarct regions immediately post MI). Five days later, the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography. The expression of TIMP-1 was measured by RT-PCR and Western blot. Collagen type I and type III were assessed by RT-PCR and immunohistochemical stain.

RESULTSThe myocardial expressions of MMP-2, MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group. Myocardial expressions of MMP-2, MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover, the expressions of collagen type I, collagen type III and the ratio of I/III collagen in border zones of infarcted myocardium were decreased by 47.6% (P < 0.01), 23.6% (P < 0.05), and 17.9% (P < 0.05) respectively, while the expression of TIMP-1 increased by 73.1%(P < 0.05) in MI/AAV2-IL-10 group compared to MI/AAV2 group.

CONCLUSIONIn vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.

Animals ; Extracellular Matrix ; metabolism ; Gene Expression ; Genetic Therapy ; Interleukin-10 ; genetics ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; genetics ; metabolism ; physiopathology ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transfection ; Ventricular Remodeling

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.

Adenocarcinoma ; blood supply ; physiopathology ; Capillaries ; metabolism ; ultrastructure ; Cell Separation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; ultrastructure ; Humans ; Microcirculation ; Stomach Neoplasms ; blood supply ; physiopathology

It has need to separate red blood cells (RBC) from marrow graft in ABO group unmatched BMT and auto-BMT with purging tumor cells, the separating effect of methylcellulose was observed. The mixture of 0.5% methylcellulose and bone marrow was laid up in an open transfusion system, and then sedimentation of RBC was performed in the transfusion tube. The separating results of 18 marrow grafts showed that the recovery rates of mononuclear cells and CD34(+) cells were (83.8 +/- 55.2)% and (90.3 +/- 7.2)%, respectively. RBC residual rate was (4.3 +/- 1.5)%. The yield of CFU-GM was (60.8 +/- 22.4)/2 x 10(5) MNC, and there was no difference to [(69.8 +/- 23.4)/2 x 10(5) MNC] yielded from same marrow samples, separated by Ficoll-Hypaque separation. It is concluded that this method could be used for bone marrow transplantation.
Bone Marrow Transplantation ; methods ; Cell Separation ; methods ; Erythrocytes ; immunology ; Humans ; Methylcellulose ; pharmacology

OBJECTIVETo explore the roles of epidermal growth factor receptor (EGFR) signaling pathway on cultured human nasal epithelial cells RPMI-2650.

METHODSRPMI-2650 cells were cultured in vitro, the growth curve was measured and the ultrastructure was observed using scanning electron microscope. When the cells were significantly confluent, they were divided into 4 groups, group A: maintained in Eagle's minimum essential media (EMEM) medium without adding any stimulators; group B: added with epidermal growth factor (EGF) 25 ng/ml; group C: added with AG1478 (EGFR selective inhibitor) 10 micromol/L followed by EGF 25 ng/ml 30 minutes later; group D: added with PD98069 (p44/42MAPK selective inhibitor) 30 micromol/L followed by EGF 25 ng/ml 30 minutes later. After incubated for 24 hours, the expression of EGFR and MUC5AC proteins in the cells of these 4 groups was studied using cytoimmunity and Western blotting.

RESULTSRPMI-2650 cells were significantly confluent after incubated for 5 to 7 days. The shape of cells was round or oval, and a large number of microvilli covered to their surface but without cilia under scanning electron microscope. The EGFR protein was expressed in the cells of group A and D, abundantly in group B, while weakly in group C. The values of comparative absorbance had significant difference between group A, B, D and group C, respectively (P < 0.01). For the MUC5AC protein, its expression was strong in the cells of group A, abundant in group B, and weak in group C and D. Significant difference of the values of comparative absorbance was analyzed between group B and group C, D, respectively (P < 0.01), while no difference between group C and group D (P > 0.05).

CONCLUSIONSThe production of MUC5AC in human nasal epithelial cells RPMI-2650 is regulated via the expression and activation of epidermal growth factor receptor signaling pathway.

Cell Proliferation ; Epithelial Cells ; metabolism ; Humans ; Mucin 5AC ; genetics ; metabolism ; Mucins ; metabolism ; Nasal Cavity ; cytology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Tumor Cells, Cultured

To study whether gene IGSF4 was inactived by methylation in leukocytic cells, expression of IGSF4 was examined before and after treatment with demethylating agent in U937, Molt4 and HL-60 leukemia cell lines by means of RT-PCR. The methylation of promoter in U937, Molt4 and HL-60 cells as well as 21 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of IGSF4 promoter was inactived and could be reversed by treatment with a demethylating agent in U937, Molt4 and HL-60 cell. IGSF4 promoter methylation was detected in 57.1% of acute leukemia patients. There is no difference in incidence of IGSF4 promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, IGSF4 is frequently inactived in acute leukemia and is a good candidate for the leukemia suppressor gene. As a normal suppressor gene, it may play an important role in inhibiting the development of leukemia, and the methylation of gene IGSF4 may be a good index in monitoring relapse of leukemia.
Acute Disease ; Cell Adhesion Molecule-1 ; Cell Adhesion Molecules ; Cell Line, Tumor ; DNA Methylation ; Humans ; Immunoglobulins ; genetics ; Leukemia ; genetics ; Membrane Proteins ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Tumor Suppressor Proteins

OBJECTIVETo observe the expression of p-p38 mitogen-activated protein kinase in lung tissues of acute lung injury rat model induced by lipopolysaccharide (LPS) and to explore the protective effects of melatonin (MT) in lung tissues in rats.

METHODSSeventy-two rats was randomly assigned to three groups, control group, LPS group and LPS + MT group. Rat model of ALI was established by instilling LPS intratracheally. We used immunohistochemical SP and Western blot method to detect the expression of p-p38 mitogen-activated protein kinase in lung tissues and used light microscope to observe morphological changes.

RESULTSThere were rare p-p38 mitogen-activated protein kinase positive cells scattered in alveolar and airway epithelial cells in control group (P < 0.01). The positive p-p38 mitogen-activated protein kinase cells in LPS group increased obviously than those in control group (P < 0.01), and were mainly distributed in infiltrative inflammatory cells, airway epithelial cells, alveolar epithelial cells and pleurames epithelial cells. In MT group, the p-p38 mitogen-activated protein kinase positive cells in airway and lung tissues were much less than those in the LPS group (P < 0.05). The Western blot results were consistent with those of immunohistochemical method.

CONCLUSIONThe expression of p-p38 mitogen-activated protein kinase increases in alveolar and airway epithelial cells in acute lung injury rat models induced by LPS. The activation of p-p38 mitogen-activated protein kinase is found in most lung tissues, suggesting that p-p38 mitogen-activated protein kinase participates in the signal transduction in inflammatory and noninflammatory cells. MT is an effective antioxidant, which relieves the inflammation in acute lung injury rats, possibly through the inhibition of the pathway of p38 MAPK over activation.

Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Lipopolysaccharides ; adverse effects ; Lung ; drug effects ; metabolism ; Male ; Melatonin ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo introduce the METRx microendoscopes diskectomy system in the treatment of far lateral lumbar disc herniation.

METHODSFourteen cases of far lateral lumbar disc herniation were operated with METRx from February 1999 to December 2002. Among them, the average age was 49 years old (range 41 - 55 years old), male in 10 cases, female in 4 cases. All cases were single disc herniation; L(4), 5 herniation in 6 discs, L(5)-S(1) herniation in 8 discs; foraminal disc herniation in 6 cases, extra-foraminal disc herniation in 8 cases.

RESULTSAll the cases were followed up from 12 to 46 months (average 26.5 months) with the results of excellence in 10 cases, good in 3 cases, fair in 1 case and no failure case. There were no disc infection, dura laceration, nerve root injury and herniation recurrence.

CONCLUSIONSMETRx is suitable for far lateral lumbar disc herniation with the advantages of minimal invasive, complete decompression of nerve root and rapid recovery. The correct approach and precise surgical technique are the key points for this operation.

Adult ; Diskectomy ; methods ; Endoscopy ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc Displacement ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Microsurgery ; Middle Aged ; Treatment Outcome