Objective:To study the influence of inserting glycines(Gly) on biological properties of HIV Tat-(Gly)_(n)-thymidine kinase(-TK) fusion proteins.Methods: Different fragments containing 0,2,4 or 6 Gly were inserted between the HIV Tat gene and TK using gene splicing by overlap extension(SOEing) PCR,and the products were cloned into PBK vector.The vectors were then transferred into E.coli after sequencing.After IPTG induction, bacilli were collected and destructed by ultrasound;the fusion protein was collected and identified by monoclonal antibody of HIV protein.HepG2 cells were incubated with DMEM supplemented with 1 ?g/ml fusion protein containing 0,2,4 or 6 Gly for 24 h.HepG2 cells of different groups were detected by immunofluorescence assay with HIV Tat monoclonal antibody;the apoptosis rate of HepG2 cells was determined by cell flow cytometry after they were incubated with gencilovir(10 ?g/ml) for 3 d and the survival rate of cells was recorded by trypan blue in different groups.Results: The recombined genes containing 0,2,4 or 6 Gly were successfully constructed,inserted into PBK vectors,and expressed into E.coli.Their proteins were obtained and purified.The level of fluorescence in different groups was similiar,but the cell survival rate and apoptosis rate were different.The highest apoptosis rate was 14.77%,which was found in the group containing 4 Gly,followed by 12.69% in 2 Gly group,8.31% in HIV Tat-TK group,4.36% in 6 Gly group,and 1.0% in group containing no Gly.Significant differences were found between each 2 groups(P

Objective To study the regulatory effect of cefodizime on the T-lymphocyte subsets of peripheral blood in mice with immunological liver injury.Methods The mouse model of immunological liver injury was induced by Bacillus Calmette Guerin and Lipoposaccharide.The study was conducted by using completely random design.The mice with immunological liver injury were divided into thymosin group,cefodizime high-,medium-,low-dose groups,ceftriaxone group and the normal saline group.The six groups were continuously administered agents respectively for 7 days,and T-lymphocyte subsets of peripheral blood in mice were determined and contrasted with those of the normal mice treated with normal saline on the 7th day.Flow cytomytry was used to determine the effects of cefodizime on T-lymphocyte subsets of peripheral blood in mice by using immuno-fluorescence technique.Results The immunological liver injury mice were deficient because their CD3+(%),CD4+(%),CD8+(%) and the ratio of CD4+CD8+ were lower than those of the normal mice.Cefodizime effectively increased CD3+(%),CD4+(%) and the ratio of CD4+CD8+ of the mice with immunological liver injury.Conclusion Cefodizime effectively improves the immune function of the host by regulating the balance between CD4+ cell and CD8+ cell.

Great changes in drug metabolizing enzyme (DME) expression occur in the fetus and child during development. Individual hepatic DME ontogeny can be categorized into one of three groups based on developmental trajectories.Some enzymes such as CYP3A7,are expressed at highest level in the fetus dur-ing the first trimester and either remain elevated or slightly de-crease during gestation,but are silenced or reduced to relatively low levels within one to two years after birth.SULT1 A1 is an ex-ample of the second group of DME.These enzymes are ex-pressed at relatively constant levels throughout gestation and into adulthood.CYP3A4 belongs to the third DME group .These en-zymes are expressed at negligible or low levels in the fetus.Sig-nificant increases in enzyme levels are exhibited within the first one to two years after birth.The epigenetic regulation refers to genomic modifications that do not involve changes in DNA se-quence and include DNA methylation,histone modifications, and non-coding RNAs.The epigenetic regulation mechanisms are responsible for the developmental expression of DME genes dur-ing liver maturation.This review will provide a summary of DME developmental expression profiles and reveal epigenetic mecha-nisms underlying variable drug metabolism and drug response. Thus,knowledge regarding DME ontogeny has permitted im-proved capability to predict drug disposition in pediatric pa-tients,which is crucial for improving drug dosing leading to opti-mal safety and efficacy in children.

Objective To investigate the effects of age on the pharmacokineties and pharmacodynamics of levobupivacaine after epidural administrstion.Methods Forty-five ASA Ⅰ or Ⅱ patients of both sexes (26 male, 19 female) aged 30-72 yr, weighing 52-83 kg scheduled for elective lower extremity surgery under epidural anesthesia, were divided into 3 age groups ( n = 15 each) : group Ⅰ≤45 yr; group Ⅱ 46-64 yr and group Ⅲ > 64 yr. Epidural anesthesia was performed at the L1,2 interspace. All of the patients received levobupivacaine 1.8 mg/kg with epinephrine 5 μg/ml given epidurally over 1.5 min. Assessment of sensory block (onset time, peak effect time, upper spread of sensory block, duration of anesthesia) and degree of motor block (using modified Bromage scale) were made. Blood samples were taken from central vein at 0, 10, 20, 30, 45, 60 90, 120, 240, 360, 480, 840 and 1 440 min after epidural administration for determination of plasma concentration of levobupivacaine by high-performance liquid chromatography (HPLG) in nine patients in each group. The pharmacokinetic parameters were calculated from plasma concentration-time data with 3P97 software package. Results The cephalad spread of sensory block was significantly higher in group Ⅲ than in group Ⅰ . The duration of sensory and motor block was significantly longer in group Ⅲ than in group Ⅰ . The plasma concentration-time curves of levobupivacaine were fitted to a two-compartment open model in the 3 groups. The plasma concentrations of levobupivacaine were significantly higher at 1 440 min after epidural administration in group Ⅲ and Ⅱ than in group Ⅰ. The t1/2β was significantly different among the 3 groups. Conclusion 0.75% levobupivacaine is safe and effective for epidural anesthesia. Age affects the pharmacokinetics (t1/2β in particular) and pharmacodynamics of levobupivacaine administered epidurally.

Objective To evaluate the correlation between CYP3A4 enzyme and analgesia with fentanyl after gynecological operation. Methods One hundred and fifty-nine ASA Ⅰ or Ⅱ patients, aged 30-50 yr, scheduled for elective myomectomy or abdominal total hysterectomy, were enrolled in this study. Anesthesia was induced with midazolam, remifentanil, propofol and succinylcholine and maintained with iv infusion of propofol and remifentanil and intermitent iv injection of atracurium. Venous blood samples were obtained for determination of the plasma 1'-hydroxymidazolam and midazolam concentrations at 1 h after iv injection of midazolam. The ratio of the 1'-hydroxymidazolam concentration to the midazolam concentration was used to reflect the effect of CYP3A4 enzyme. Pain was assessed with visual analog scale (VAS) after consciousness was regained. When VAS score ＞ 4,the patients were given fentanyl 10 μg every 5 min until VAS score ≤ 4 and then PCIA with fentanyl was performed. VAS score was maintained ≤4. The times of successful delivery within 24 h after operation and during the period of 24-28 h after operation and fentanyl consumption within 48 h after operation were recorded. Pearson correlation was used to analyze the data. Results There was no correlation between the effect of CYP3 A4 enzyme and the times of successful delivery or fentanyl consumption, and the correlation coefficients were 0.16, 0.13 and 0.11 respectively ( P ＞ 0.05). Conclusion CYP3A4 enzyme is not the major enzyme metabolizing fentanyl.

Objective To investigate the pharmacokinetics of different concentrations of levobupivacaine for lumbar epidural anesthesia.Methods Twenty ASA Ⅰ or Ⅱ patients of both sexes, aged 35-59 years and scheduled for elective radical resection of rectal or colon carcinoma under general anesthesia combined with epidural block, were randomly divided into 2 groups (n=10 each):group Ⅰ (receiving 0.75% levobupivacaine) and group Ⅱ (receiving 0.5% levobupivacaine). Epidural block was performed at L1-2 interspace. Group Ⅰ and Ⅱ received epidural 0.75% and 0.5% levobupivacaine 2 mg/kg (containing adrenaline 5 μg/kg)injected slowly over 2 min, respectively. And 30 min later, general anesthesia was induced with y-hydroxybutyrate 60-80 mg/kg and remifentanil 1-2μg/kg. Tracheal intubation was facilitated with succinylcholine 1-1.5 mg/kg and the patients were mechanically ventilated. Anesthesia was maintained with inhalation of nitrous oxide (N2 O) and O2 (1:1) and continuous infusion of remifentanil 0.01-0.1μg·kg-1·min-1 and intermittent intravenous boluses of atracurium. Sensory and motor blocks were assessed after epidural levobupivacaine. Blood samples were taken from the central vein at 0, 10, 20, 30, 45, 60, 90, 120, 210, 300, 420,540, 660 and 840 min, respectively, after epidural administration for determination of plasma concentrations of levobupivacaine by high performance liquid chromatography.Results The plasma concentration-time curves of levobupivacaine were fitted to a two-compartment open model in the two groups and there were no significant differences in the pharmacokinetic profiles between the two groups. The onset time of sensory and motor blocks was shorter and the duration of the two blocks was longer with 0.75% levobupivacaine as compared with 0.5%levobupivacaine. The incidences of nausea and vomiting and hypotension were low and no severe cardiovascular and neurological side-effects developed.Conclusion The pharmacokinetic parameters do not differ significantly between epidural 0.75% and 0.5% levobupivacaine when the total doses are the same. And epidural anesthesia with either 0.75% or 0.5% levobupivacaine is safe.

Objective To investigate the effect of acute hypervolemic hemodilufion (AHHD) on EC50 of propofol by target-controlled infusion (TCI) for loss of consciousness (LOC). Methods Sixty ASA Ⅰ or Ⅱ patients aged 18-64 yr scheduled for vertebral eolunm or total hip replacement surgery were randomly divided into 4 groups (n = 15 each) : group Ⅰ target plasma concentration of propofol (Tp) ;group Ⅱ target effect-site concentration of propofol (Te) ;group Ⅲ AHHD + Tp and group Ⅳ AHHD + Te. All the patients recoived iv infusion of lactated Ringer's solution 0.7 ml·kg-1 ·h-1 via peripheral vein for 30 min. At the same time 4% gelofusion 15 ml/kg was infused over 30 min via internal jugular vein in group Ⅲ and Ⅳ. At the end of gelofusine infusion TCI of propofol was started. The initial target concentration was set at 1.2 μg/ml. After the target concentration was steadily maintained for 30 s, the consciousness of the patients was evaluated by an anesthesiologist not involved in the study using OAA/S scale. The target concentration was increased in 0.3 μg/ml increment until the patients lost consciousness (OAA/S = 0). The target plasma concentration and effect-site concentration were then recorded. EC50 and 95% confidence interval (CI) of propofol for LOC were calculated by probit analysis. Results The ECho (95 % CI) of propofol for patients in group Tp, Te, AHHD + Tp and AHHD + Te (group Ⅰ-Ⅳ) were 3.74 (3.46-4.16), 2.32 (2.17-2.42), 4.12 (3.81-4.32) and 2.38 (2.14-2.56) μg/ml respectively. EC50 was significantly higher for loss of consciousness in AHHD + Tp group (group Ⅲ)than in Tpgroup (group Ⅰ), but there was no significant difference in EC50 between group Te and group AHHD + Te. Conclusion AHHD can increase the EC50 of target plasma concentration of propofol by TCI for LOC but has no effect on EC50 of target effect-site concentration.

BACKGROUND:Adipose-derived stem cels are regarded as the potential seed cels for tissue engineering. Colagenase digestion is used to isolate adipose-derived stem cels from fat pads currently. However, there are some problems, such as cumbersome operation and high cost.
OBJECTIVE: To study the basic biological characteristics of adipose-derived stem cels by tissue explants culture and to explore the differentiation potential into osteoblasts, adipocytes and endothelial progenitor cels in vitro.
METHODS:Adipose-derived stem cels were isolated by tissue explants technique from the bilateral groin fat pads of rats under aseptic conditions, and cultured in vitro. Cel counting kit-8 was used to detect the proliferative activity, and flow cytometry was employed to analyze the expression of cel surface markers. Passage 4 adipose-derived stem cels were cultured in osteogenic medium, adipogenic medium and endothelial progenitor cel medium for 2-3 weeks, and then the cels were identified.
RESULTS AND CONCLUSION:Adipose-derived stem cels that were isolated by tissue explants culture were easily cultured, and after subculture, cels were mainly spindle-shaped and grew in clone-like manner with swirling arrangement. Cels that experienced repeated subcultures stil kept stronger proliferative ability and the cel growth curve was shaped as a parabola. Immunochemical staining analysis revealed that adipose-derived stem cels were positive for CD44, CD90 and CD29, but negative for CD31, CD45. After adipogenic/osteogenic induction, the cels were respectively positive for oil red O staining and alizarin red staining. Induced endothelial progenitor cels were identified with CD34 and the ability to uptake Dil-ac-LDL and FITC-UEA. These findings indicate using the using tissue explants culture, high-purity adipose-derived stem cels easy to proliferate can be harvested, highly express stem cels-related antigens, and have the ability to differentiate into osteoblasts, adipocytes and endothelial progenitor cels, which meet the needs of seed cels in tissue engineering research.

Objective To evaluate the changes in the expression of NRF-1 in the spinal cord during remifentail-induced hyperalgesia in a rat model of incisional pain.Methods Forty-eight Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 4 groups (n =12 each) using a random number table:control group (C); incisional pain group (group Ⅰ); remifentanil group (group R); incisional pain + remifentanil group (group Ⅰ + R).All the rats were anesthetized with sevoflurane.A 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the right hindpaw in I and I + R groups.In C and I groups,normal saline was subcutaneously infused for 30 min.In group I + R,remifentanil (0.04 mg/kg,0.4ml) was subcutaneously infused for 30 min starting from the onset of skin incision.Paw withdrawal threshold to mechanical stimulation (PWMT) was measured at 24 h before operation and at 2,6,12,24 and 48 h after operation.After measurement of PWMT at 48 h,the rats were sacrificed and L4,5 segments of the spinal cord were removed rapidly to detect the expression of nuclear respiratory factor 1 (NRF-1) by immunofluorescence and Western blot.Results Compared with group C,PWMT was significantly decreased at each time point after operation,and the expression of NRF-1 in the spinal cord was up-regulated in I and I + R groups (P ＜ 0.05).Compared with group I,PWMT was significantly decreased at each time point after operation,and the expression of NRF-1 in the spinal cord was up-regulated in group I + R (P ＜ 0.05).Conclusion Up-regulation of NRF-1 expression in the spinal cord may be involved in the development of remifentail-induced hyperalgesia in a rat model of incisional pain.

Objective To evaluate the effects of gender on the pharmacokinetics of propofol in patients.Methods Twenty ASA physical status Ⅰ or Ⅱ patients of both sexes (10 male,10 female),aged 42-59 yr,weighing 46-76 kg,scheduled for elective surgery for gastrointestinal cancer,were randomly divided into 2 gender groups (n =10 each):male group and female group.Combined intravenous-inhalational anesthesia was performed during surgery.Anesthesia was induced with iv injection of 2％ propofol 2 mg/kg,remifentanil 1.5 μg/kg,and suxamethonium chloride 1.5 mg/kg.The patients were tracheally intubated and mechanically ventilated.Anesthesia was maintained with inhalation of the mixture of 1％-2 ％ isoflurane and nitrous oxide (N2 O ∶ O2 =1 ∶ 1.),iv infusion of remifentanil 0.2-0.3 μg/kg and intermittent iv boluses of atracurium.Blood samples were taken from the central vein before propofol administration,and at 1,2,3,4,6,10,15,30,45,60,90,120,180,240,360 and 720 min after propofol administration for determination of the plasma concentration of propofol by high-performance liquid chromatography.The blood concentration-time curve of propofol was drawn and the pharmacokinetic parameters were calculated.Results The blood concentrations of propofol were significantly lower at each time points within 10 rmin after administration of the single bolus of propofol in female group than in male group (P ＜0.05).The blood concentration-time curves of propofol were fitted to a three-compartment open model in the 2 groups.The central volume of distribution and clearance rate were significantly larger in female patients than in male patients (P ＜ 0.05).Conclusion After iv injection of propofol,the blood concentration of propofol is lower,and the central volume of distribution and clearance rate are larger in female than in male,suggesting that gender has significant effect on pharmacokinetics of propofol.