AIMTo explore the characteristic of ATP-activated current in trigeminal ganglion (TG) neurons of rat.

METHODSWhole-cell patch-clamp was performed.

RESULTS(1) The majority (92.1%) of TG neurons responded to ATP applied externally with inward currents. We recorded three distinct ATP-activated currents: fast, slow and intermediate, which were concentration-dependent. (2) In general, the fast ATP-activated currents were distributed mainly in small-diameter TG neurons, the slow ATP-activated currents were distributed mainly in large-diameter TG neurons, and the intermediate ATP-activated currents were distributed mainly in intermediate-diameter TG neurons. (3) The time course of rising phase from 10% to 90% of the three distinct ATP-activated currents were as follows: fast: (33.6 +/- 4.5) ms; intermediate: (62.2 +/- 9.9) ms; slow: (302.1 +/- 62.0) ms, and that of desensitizing phase were (399.4 +/- 58.2) ms (fast), and > 500 ms (slow) respectively. (4) From the current-voltage relationship curves, it can be seen that the reversal potential values of the three distinct ATP-activated currents were the same, all being 0-5mV. And they all were characterized by inward rectification. (5) The dose-response curve for fast ATP-activated current shifted downwards as compared with the intermediate ATP-activated current, and that for the slow ATP-activated current shifted upwards.

CONCLUSIONThe EC50s of the three curves tended to be identical. The results suggested that three kinds of distinct ATP-activated currents could be mediated by various subtypes of P2X receptors assembled by different subunits, and the subtypes existed in TG neurons of different diameters and transmit different information.

Animals ; Cells, Cultured ; Membrane Potentials ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2 ; metabolism ; Trigeminal Ganglion ; physiology

OBJECTIVETo explore the feasibility of using a nonreactive, permanent endoskeletal scaffold to create the prothesis in special shape which is covered with tissue-engineered cartilage.

METHODSPorcine BMSCs and articular chondrocytes were isolated and expanded respectively in vitro. Porcine BMSC of passage 1 in the concentration of 10 x 10(7)/ml were seeded onto a cylinder-shaped PGA (1 mm in thickness)/Medpor (3mm in diameter and 5mm in highness) scaffold as the experimental group. After the cell-scaffold constructs were cultured for 5 days, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium for 4 weeks. BMSCs and chondrocytes of the same concentration were seeded respectively onto the scaffold as the negative control group and the positive control group. After cultured in vitro for 4 weeks, the cell-scaffolds construct were implanted into subcutaneous pockets on the back of nude mice. Four and eight weeks later, the formed cartilage prosthesis were harvested and then evaluated by gross view, histology, immunohistochemistry and glycosamino-glycan (GAG) content.

RESULTSCells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. All specimens in experimental group and positive control group formed mature cartilage with collagen II expression.The mature catrtilage wraped HDPE compactly and grown into the gap of HDPE. Mature lacuna structures and metachromatic matrices were also observed in these specimens. GAG contents in experimental group were (5.13 +/- 0.32) mg/g (4 weeks), (5.37 +/- 0.12) mg/g (8 weeks). In contrast, specimens in BMSC group showed mainly fibrous tissue.

CONCLUSIONIt indicates that it is feasible to create special shaped tissue-engineering cartilage with the permanent internal support using BMSCs as seed cell.

Animals ; Bone Marrow Cells ; cytology ; Cartilage ; cytology ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Mice ; Mice, Nude ; Stromal Cells ; cytology ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

AIMTo study the correlation between 5-HT-induced pain response and the contribution by individual 5-HTR subtypes including 5-HT1R, 5-HT2R and 5-HT3R at the level of peripheral primary afferent.

METHODSThe experiments were done on acutely isolated trigeminal ganglion (TG) neurons using whole-cell patch clamp technique and the nociceptive effect was observed on behavior experiments by intraplantar injection of test drugs.

RESULTSThe majority of cells examined responded to 5-HT in a manner of concentration dependence (10(-6) - 10(-3) mol/) (61.4%, 54/88) and with a fast activating and rapid desensitizing inward current (I(5-HT)), which was thought to be mediated by the activation of 5-HT3R, since it could be blocked by 5-HT3R antagonist ICS 205930 and mimicked by 5-HT3R agonist 2-methyl-5-HT. It was found that I(5-HT) was potentiated by 5-HT2R agonist alpha-methyl-5-HT markedly, while 5-HT1R agonist R-(+)-UH 301 did not. In behavioral experiment performed on conscious rats, intraplantar injection of 5-HT(10(-5), 10(-4) and 10(-3) mol/L) induced an increment of cumulative lifting time first 20 min in a manner of concentration dependence. By dissociating 5-HTR subtypes using their corresponding antagonists (ICS and CYP) the potency order of hindpaw lifting time was identified as follows: 5-HT > 5-HT + ICS > 5-HT + CYP.

CONCLUSIONThe results suggest that in 5-HT-induced nociceptive response at the primary sensory level 5-HT3R may play a role of initiation, but 5-HT2R mediates maintaining and modulatory effect in the processes of nociceptive information convey.

Animals ; Male ; Membrane Potentials ; Pain ; physiopathology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin, 5-HT1 ; metabolism ; Receptors, Serotonin, 5-HT2 ; metabolism ; Receptors, Serotonin, 5-HT3 ; metabolism ; Sensory Receptor Cells ; metabolism ; physiology

OBJECTIVETo evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms.

METHODSFluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9.

RESULTSMetformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin.

CONCLUSIONMetformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.

Apoptosis ; drug effects ; Carcinoma, Renal Cell ; pathology ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Kidney Neoplasms ; pathology ; Metformin ; pharmacology

OBJECTIVETo investigate the relationship between hepatitis C virus (HCV) genotype, serum viral load and ALT levels, and the factors associated with the viral relapse after IFN treatment in patients with chronic hepatitis C.

METHODSThe HCV RNA levels were determined with Cobas Amplicor Monitor Test, version 2.0, and HCV genotypes were examined by means of PCR products of 5' NTR digested with restriction endonucleases. The patients with chronic hepatitis C were treated with PEG-IFN alpha -2a and Roferon-A for 24 weeks. Those with a viral response after 24 week treatment were followed for an additional 24 weeks. The association of clinical characteristics, such as sex, age, the way of the HCV infection, IFN treatment history and platelet counts, and the HCV genotype, virus load and medicine used for the viral relapse after IFN treatment were analyzed.

RESULTSOf the 208 chronic hepatitis C patients, the ALT levels were not related to HCV RNA levels (r = 0.093, P > 0.05). No difference of ALT levels between HCV genotypes was found, and the HCV RNA load was also of no difference between HCV genotype 1 patients and non 1 patients. Of the 119 patients with viral response after 24 week treatment, 58 cases (48.7%) relapsed after another 24 week's follow-up. Relapse was not significantly related to the clinical characteristics, such as sex, age, mode of the infection, treatment history of IFN, AST/ALT ratio, platelet counts and the baseline viral load. Among patients with genotype 1 virus, the relapse rate was significantly higher than those patients with non-genotype 1 virus (54.5% vs 32.1%, P=0.039). The relapse rate after PEG-IFN alpha -2a treatment was lower than that of Roferon-A treatment (47.0% vs. 52.8%), but not significantly.

CONCLUSIONThe viral relapse of chronic hepatitis C patients after IFN treatment was significantly associated with the genotypes of the HCV.

Antiviral Agents ; therapeutic use ; Female ; Genotype ; Hepacivirus ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; RNA, Viral ; blood ; Recombinant Proteins ; Recurrence ; Treatment Outcome ; Viral Load

Objective To explore the responses and mechanisms of peripheral primary afferent neurons to adenosine 5'-triphosphate (ATP) and bradykinin (BK) applied separately or in combination by electrophysiological recording and behavioral observation. Methods The experiments were done on samples of acutely isolated rat dorsal root ganglion (DRG) neurons by the whole-cell patch clamp recording technique, to record ATP-activated current (IATP) and the regulating effect of BK on IATP and to observe the global behavior with pain behavioral experiment. Results ATP added after the pretreatment of BK in the majority of detected cells, IATP would be reinforced significantly, the degree of increment depending on the concentration of BK (BK 10-6 -10-4 mol/L), while the EC50 values of the concentration-response curve with and without pretreatment of BK were very close to each other (1.65×10-5 mol/L vs 2.0×10-5 mol/L). In the behavioral experiment, subcutaneously intraplantar injection of BK and ATP separately in hind limbs of rats both induced concentration-dependent pain behavioral (paw lifting) responses, while the duration of hindpaw lifting was prolonged dramatically with the increase in the ATP concentration, when BK (10-6 mol/L) was injected in combination with ATP (10-5, 10-4 and 10-3 mol/L). Conclusion Inflammatory mediators like BK and ATP etc play an important role in the production, transmission and modulation of pain information in peripheral sensory nerve endings. Both electrophysiological and behavioral experiments demonstrate that there is a synergic effect between ATP and BK, which is thought to be non-competitive. BK may reinforce IATP remarkably, and the pain responses induced by the increment in ATP concentration increase with the existence of BK.