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Objective To construct and identify adenovirag (Ad-RUNEP) carrying human B-endorphin(B-EP) genes which can be regulated by mifepfistone (RU486)-inducible system and to evaluate the effects of different concentrations of RU486 on the transgene expression in adenovirus in vitro.Methods The shuttle plagmid pDC312一RUNEP carrying B-EP genes which can be regulated by RU486-inducible system was constructed and was combined with adenovims to form a recombinant Ad-RUNEP using AdMAXTM system.The recombinant Ad.RUNEP was then amplified and purified.The titers of the adenovirus were determined and the adenovirus vector was checked.After being infected by Ad-RUNEP for 24 h,A431 cell line was incubated in liquid culture media containing RU4860,10-10,10-9,10-8,10-7,and 10-6 mol/L respectively(group R0-5) for 48h,and Was then transferred to liquid culture media containing no RU486.The liquid culture media were obtained on 1st.2nd and 4th day of incubation and centrifuged.The supematant Was collected for determination of B-EP concentration by ELISA.Results The analysis of enzyme-incision demonstrated that RU486 regulating system and B-EP were cloned directly into pDC312-RUNEP.The titer of Ad-RUNEP was 2.25×1010 pfuml.The expression of B-EP was significantly higher in group R1-s than in group R0 and was significantly lower in group R1-3 than in group R4 (P<O.05).The expression of B-EP wag significantly higher on 2nd than on 1st and 4th day.Conclusion The adenovims carrying B-EP genes which can be regulated by RU486 (Ad-RUNEP) Wag successfully constructed.
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Objective To determine if propofol-remifentanil and isoflurane have any different effects on intrapulmonary shunt during one-lung ventilation (OLV). Methods Twenty-four ASA Ⅰ or Ⅱ patients (18 male, 6 female) aged 42-69 yr undergoing radical esophagus cancer resection via left thoracotomy were randomly divided into 2 equal groups ( n = 8 each) : propofol group (Pro) and isoflurane group (Iso) . The preoperative lung function was normal in both groups. The patients were premedicated with intramuscular diazepam 10 mg and atropine 0.5 mg. Radial artery was cannulated and S-G catheter was placed via right internal jugular vein in pulmonary artery. Anesthesia was induced with propofol 1.5-2.0 mg?kg-1, fentanyl 4 ?g?kg-1 and succinylcholine 1.5 mg?kg-1 and maintained with TCI of propofol and remifentanil (target plasma concentration was set at 3.2 ?g?ml-1 and 4.5 ng?ml-1 ) or isoflurane inhalation ( end- tidal isoflurane concentration = 1.5% -2.5 % ) and intermittent i. v. boluses of fentanyl. The patients were mechanically ventilated after endobronchial intubation with double-lumen tube (VT = 8-10 ml, RR= 10-12 bpm, I:E = 1:2) . During OLV VT was reduced to 6-8 ml and RR increased to 14-16 bpm.PaCO2 was maintained at 35-45 mm Hg. ECG, HR, MAP, SpO2, auditory-evoked potential index (AAI), cardiac index (CI), airway pressure and end-tidal isoflurane concentration were continuously monitored during operation. Blood samples were taken from radial artery and pulmonary artery at 10 min after S-G catheter placement (T0, baseline) at 10 min bilateral ventilation (in right lateral position) (T1) at 15, 30, 60, 90 min of OLV (T2-5) for measurement of blood gases and calculation of Qs/Qt.Results The two groups were comparable with respect to age, M/F ratio, body weight and preoperative lung function. AAI was below 30 during operation and PaCO2 and pH were within normal range in both groups. Qs/Qt was significantly increased while PaO2 was significantly decreased during operation (from T1 to T5 ) as compared to baseline values (T0) but Qs/Qt was gradually decreasing while PaO2 was gradually increasing from T2-5 in both groups. Qs/Qt was significantly lower in Pro group than in Iso group at each interval (T2-5) but there was no significant difference in PaO2 at T2-5 between the two groups. Conclusion There is less intrapulmonary shunt during OLV under general anesthesia with propofol-remifentanil than with isoflurane but the difference is not significant enough to affect PaO2.
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Objective To determine whether thiol can affect the activity of nuclear factor kappa B (NF-?B) in and release of TNF-?from lipopolysaccharide (LPS)-activated kupffer cells (KCs) and ascertain whether these effects are mediated through glutathione (GSH) .Methods KCs were isolated from livers of healthy male adult SD rats weighing 250-300g and cultured for 12h. The cultured KCs were exposed to LPS 10 ng?ml-1. The effects of different concentrations of glutathione monoethylester (GSHEE) and N-acetylcysteine (NAC) on NF-?B activity in and release of TNF-afrom KCs were determined. The effect of pretreatment with GSH synthesis inhibitor - BSO on the inhibitory effect of NAC on inflammation was investigated. NF?B activity was determined by electrophoretic mobility shift assay (EMSA) and intracellular GSH content and TNF-?level in supernatant by HPLC. Results Intracellular GSH content remained unchanged after exposure of KCs to LPS. Both GSHEE and NAC increased intracellular GSH level and inhibited NF-?B activity and release of TNF-?. BSO blocked the increase in GSH induced by NAC but did not affect the inhibitory effect of NAC on TNF?release. Conclusions LPS does not affect GSH level in KCs. NAC can inhibit NF-?B activity in and TNF-?release from KCs and increase intracellular GSH level but the inhibitory effect is not mediated through GSH.
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Objective To examine the analgesic effect of intrathecal (i.t.) adenovirus containing human beta-endorphin (?-EP) gene in a rat model of neuropathic pain produced by chronic constrictive injury (CCI) . Methods Thirty-six male SD rats weighing 210-260 g were randomly divided into 3 groups: (1) blank control group (n = 5); (2) sham-operated group (n = 5) ; (3) neuropathic pain group (n = 26) . The neuropathic pain group was further divided into 3 subgroups: Ad-NEP subgrouop (n = 9); Ad-GFP (the recombinant adenovirus containing green fluorescent protein) subgroup (n = 9) and normal saline (NS) subgroup (n = 8). The animals were anesthetized with intraperitoneal ketamine 100 mg?kg-1 and atropine 50 mg?kg-1. Neuropathic pain was produced by ligation of right sciatic nerve according to the technique described by Bennet and Xie. In sham-operated group the sciatic nerve was exposed but not ligated. In blank control group no operation was performed. Seven days after the surgery a PE-10 catheter was placed in the subarachnoid space at L5,6 according to the method of Milligan et al. Seven days after catheter placement 1?108 pfu Ad-NEP, Ad-GFP and 0.9% saline were injected i.t. via the catheter respectively. The paw-withdrawal latency to radiant heat was measured before surgery (T0,baseline) , the day of i.t. injection (T1) and 1 day (T2) , 1 w (T3), 2 w (T4), 3 w (T5), 4 w (T6) 5 w (T7) after i.t. injection. At one week after i.t. administration one animal in Ad-NEP subgroup and Ad-GFP subgroup was killed and the lumbar segment (L3-6) of the spinal cord was removed for immuno-histochemical examination. Naloxone 1 mg?kg-1 was given intraperitoneally in Ad-NEP subgroup (n = 8) and Ad-GFP subgroup (n = 8) at 10 days after i.t. injection. Pain threshold to thermal stimulation of the right paw was measured before (t0) and from 10-90 min after intraperitoneal naloxone injection at an interval of 10 min (t1-9). CSF samples were obtained at T1-7 for determination of CSF concentration of ?-EP using radio-immunological assay. Results The right paw-withdrawal latencies (PWLs) were significantly lower in Ad-GFP subgroup and NS subgroup than in blank control and sham-operated groups (P