Objective To establish an immortalized rat astrocyte strain(IAST) expressing enkephalin regulated by doxycycline(Dox) and observe its analysesic effect on rat chronic neuropathic pain. Methods Retrovirus infection method was employed to develop an immortalized rat astrocyte strain expression enkephalin regulated by doxycycline.hPPE gene expression level of IAST/Tet-On/hPPE strain was detected by Real time-PCR and radioimmunoassay.Its analgesic potential was investigated by mechanical paw withdrawal thresholds after these cells were implanted into the subarachnoid space of chronic constrictive injury(CCI) rats.The expression of Fos protein in the dorsal horn of spinal cord was determined by immunohistochemistry. Results An immortalized rat astrocyte strain secreting enkephalin under the control of doxycycline was established successfully.After transplantation of IAST/Tet-On/hPPE cell into the subarachnoid space of chronic constrictive injury(CCI) rats, the sensitivity of mechanical allodynia and the expression of Fos protein were significantly decreased(P

Objective: To compare effects of sufentanil with fentanyl when combined with propofol in intravenous anesthesia. Method: Forty ASA grade Ⅰ-Ⅱ patients, scheduled for abdominal operations, were randomly divided into two groups. Group sufentanil(Suf): combining sufentanil with propofol; group fentnyl (F): combining fentanyl with propofol. The items observed were perioperative hemodynamics, plasma epinephrine and norepinephine levels and analepsia time. Result: In group Suf, SP, DP, HR and RPP were decreased significantly (P0.05). In group F, the values above were also significantly decreased (P

ve To assess the protective effect of fentanyl on primary cultured myocardial cells against injury induced by anoxia-reoxygenation. Methods Ventricular myocardial cells enzymatically isolated and cultured in 1640 culture medium for 6 days were randomly divided into 5 groups: group A received no anoxia served as control; group B received 2 hours anoxia followed by half an hour reoxygenation; and group C, D and E received 10ng ? ml-1, 30 ng ? ml-1 and 50 ng? ml-1 fentanyl respectively before anoxia-reoxygenation. The cell viability, myocardial intracellular content of malondialdehyde (MDA) and the activities of lactic dehydrogenase (LDH) and creatine kinase(CK) were measured at the end of the experiment. Results Anoxia-reoxygenation caused dramatic decrease in cell viability, and increases in myocardial intracellular MDA content and the LDH and CK activities as compared with those in control group. Fentanyl 30 ng?ml-1 and 50 ng?ml-1 significantly attenuated the increases in LDH, CK activities and MDA content, and decrease in cell viability caused by anoxia-reoxygenation. Fentanyl 10ng?ml-1 did not produce any significant changes. Conclusions Fentanyl can produce protective effects on primary cultured cardiomyocytes against anoxia-reoxygenation injury.

Objective To investigate the effect of isovolumic hemodilution with 6% hydroxyethyl starch (HES) (200/0.5) on expression of intercellular adhesion molecule-1 (ICAM-1) after global cerebral ischemia-reperfusion in rats. Methods Eighty-four male Wistar rats weighing 230-280g were randomly divided into 3 groups: group Ⅱ sham operation (S , n = 20); group ischemia-reperfusion (Ⅱ , n = 32) and groupⅢ hemodilution with 6% HES (200/0.05) (H, n =32) . Group Ⅱ and group Ⅲ were further divided into 4 equal subgroups with 8 animals in each subgroup: 2h, 4h, 8h and 12h after beginning of reperfusion. Global cerebral ischemia was produced by permanent occlusion of bilateral vertebral arteries and cross-clamping of bilateral common carotid arteries for 10min and then clamping was released for reperfusion. In group Ⅲ acute normal volumic hemodilution was performed at 10 min after reperfusion was begun. 1ml/100g of blood was removed from artery and equal volume of 6% HES(200/0.5) was infused into the vein simultaneously. Hct was checked before and after hemodilution. The animals were decapitated at designed time and brain tissue was removed from ischemic area and frozen in liquid nitrogen. ICAM-1 expression was determined by using immunohistochemical technique. Results ICAM-1 expression significantly increased after 2h, 4h, 8h and 12h reperfusion in group Ⅱ and Ⅲ as compared with in group Ⅰ (P

Objective To investigate the role of ca2+/calmodulin-dependent protein kinase II(CaMKII)in chronic inflammatory pain in mice.Methods Forty healthy male ICR mice weighing 20-25 g were randomly divided into 5 groups(n=8 each):group A control;group B chronic inflammation;group C KN92:group D KN93 30 nmol and group E KN93 45 nmo1.Chronic inflammatory pain was produced by injecting complete Freund's adjuvant(CFA) 20 ul into the dorsal surface of left hind paw in group B,C,D and E.In group A normal saline 20 ul was injected instead of CFA.On the 1st and 3rd day after CFA injection group D,E and Creceived intrathecal administration of specific CaMK Ⅱ inhibitor KN93 30 and 45 nmol and KN92 45 nmol(aninactive analog of KN93).in 5 ul respectively.Mechanical and thermal pain threshold to von Frey hair and radiantheat stimulation were determined at 30 min before CFA injection (To,baseline),30 min before(T1) and 30 min after IT.administration (T2) on the 1st day after CFA injection and at 30 min after IT administration(T3) on the 3rd day after CFA injection.The mice were immediately killed by C02 anesthesia after the last determination of pain threshold.The lumbar segment of the spinal cord Was then removed for determination of the expression of p-CaMK Ⅱ a by Western blotting.Results The thermal and mechanical pain threshold Was significantly decreasedat T1-3 in group B,C and D but only at T1 in group E as compared with the baseline values and group A.Thermal and mechanical pain threshold were significantly hisher at T2.3 in group E than in group B.p-CaMKⅡa expression in the spinal cord was significantly higher in group B,C and D than in group A but there was no significant difference in p-CaMKⅡa between group A and E.The p-CaMKII a expression in the spinal cord was significantyly lower in group E than in group B.Conclusion CaMK Ⅱ is involved in the development of chronic inflammatory pain.

Objective To immortalize rat astrocytes which could be used as cell carriers for transgenic cellular analgesia. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats by trypsin digestion and cultured according to the method of differential cell adhesiveness and transfected with plasmid pCMVSV40T/ PUR containing the simian virus 40 large tumor antigen (SV40Tag) gene. The positive colonies were isolated by puromycin selection and expanded by many passages. The integration and expression of large T antigen gene were detected and the immuno-reactivity of glial fibrillary acidic protein (GFAP) was determined by PCR, RT-PCR and immuno-cytochemistry. Results Rat astrocytes were successfully cultured in vitro and positively stained for the astrocytic marker GFAP. The positive colonies were isolated and subcultured for 50 passages. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis. The size of amplifacation product of target gene was identical to that of the positive control (558 bp) . There was no PCR and RT-PCR product from non-transfected cells. DNA sequencing and BLAST showed that 558 bp nucleotides were identical to the SV40Tag gene of the Genbank (100%). The transfected cells were positively stained for the SV40Tag and GFAP. Conclusion Immortalized rat astrocyte strain with SV40 tag gene is constructed successfully.

Objective To study the biological character of rat astrocyte strain immortalized by simian virus 40 large T antigen gene(SV40Tag) and explore the feasibility of using it as cell vehicle for transgenic cellular analgesia.Methods Rat cerebral cortical astrocytes (AST) and immortalized rat astrocyte strain (IAST) were cultured in vitro. Morphology and growth features of AST and IAST were examined and compared after subculture, freezing and recovery. The ultrastructure of the cells was observed by transmission electron microscopy. The glial fibrillary acidic protein (GFAP) in these cells was detected by immuno-cytochemistry. The cell proliferation rate and cell cycle were determined by bromodeoxyuridine labelling and flow cytometry. AST and IAST were cultured in soft agar and inoculated in nude mice to investigate the tumorigenesis of IAST. Results LAST could be subcultured successively. The cells remained monolayer, anchorage dependent and the growth was attachment-inhibited. When AST was subcultured for only 10 passages, replicative senescense began. Subculture, freezing and recovery did not influence the shape and proliferation of IAST (94%?5%) , but decreased the vitality rate of AST (54%?4% ) (P

Objective To construct a recombinant retroviral vector with human preproenkephalin gene regulated by tetracycline. Methods Human preproenkephalin gene was amplificated by polymerase chain reaction (PCR) and was cloned into retrovirus tetracycline responsive plasmid. Then this recombinant plasmid and regulatory plasmid pRev Tet-On were transferred into packaging cell PT67 respectively. The transfected PT67 / Tet-On and PT 67 / TREhPPE cells were selected by the corresponding antibiotics and identified by RT-PCR. The selected cells were enriched and virus liter was assayed using NIH3T3. Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant RevTRE/hPPE vector was constructed successfully. The virus liter of PT 67/TREhPPE was 3.2 ? 103 CFU?ml-1 and the virus titer of PT67/Tet-On was 2.6?105 CFU?ml-1.Conclusion A recombinant retroviral vector with human preproenkephalin gene regulated by trtracycline and stable virus producing lines have been successfully constructed, providing a good basis for further research on regulated cell therapy of chronic pain.

Objective To determine the mimic epitope of N-methyl-D-aspartate receptor 2B subunit (NR2B). Methods The monoclonal antibody interacting with NR2B was used as target protein to screen the binding peptide from a 12-mer Ph. D random peptide library. After three rounds of affinity screening, twelve specific clones were selected randomly and identified by sandwich ELISA. The peptide sequences were analyzed by DNA sequencing. The clones containing a common sequence were named positive clone. The competitive inhibition of the native antigen bound to monoclonal antibody against the NR2B by the positive clone was assayed by cell ELISA. Results After 3 rounds of screening the phages specifically bound with mAb NR2B were selected and amplified. Nine of the 12 clones displayed a common aminoacid sequence: SHPPVMPWPTST. The inhibitory assay showed that the mimic epitope peptides displayed on the phage surface could effectively inhibit the combination of the monoclonal antibody with native antigen. The inhibitory rate of mimic epitope was (45?3)% .Conclusion The mimic epitope of NR2B was screened successfully from the 12-mer Ph D random peptide library. This peptide mimics an epitope of the native NR2B.

0.05) and group TP2 ( P