Objective To observe the changes of blood fentanyl concentration in rats during anhepatic phase by treatment of large dose of dexamethasone and to investigate the extrahepatic metabolism of fentanyl. Methods Thirty rats were randomly divided into 3 groups (n=10 in each group): normal control group (Group A), anhepatic phase group (Group B), anhepatic phase+dexamethasone treatment group (Group C). The hepatic portal of rats was dissociated and closed in Group B. In Group C, 20 mg/kg of dexamethasone was infused 1 h before the occlusion of hepatic portal. After intravenous injection of 20 ?g/kg fentanyl each time, the blood samples were collected. The fentanyl blood concentration was analyzed by HPLC. Results The blood fentanyl concentration of anhepatic phase dropped more slowly in Group B than Group C (P0.05). Conclusion The liver is the main organ for fentanyl metabolism. There is the extrahepatic metabolism for fentanyl and dexamethasone can enhance the extrahepatic metabolism of fentanyl.

Humans ; Medical Waste Disposal ; Occupational Health

Objective To detect the sperm apoptosis rate and level of reactive oxygen species (ROS) in seminal plasma and explore their correlation among infertile males. Methods Ninety-two inferitile males were divided into varicocele (VC) group (n=32), leukocytospermia group(n=30) and the other cause group (n=30), and another 24 in vitro fertilization sperm samples were sereved as controls. The routine sperm parameters including seminal pH, sperm viability and sperm density were examined by computer assisted sperm analysis, the sperm apoptosis rate was asseseed using Annexin V/PI staining, and the ROS level in seminal plasma was detected by TBA method. The differences in seminal parameters between three infertile groups and control group were compared, and the correlation of sperm apoptosis rate with level of ROS in seminal plasma was explored in each group. Results The sperm viability of three infertile groups was significantly lower than that of control group (P<0.01). The sperm apoptosis rates and levels of ROS in seminal plasma in VC group and leukocytospermia group were significantly higher than those in control group (P < 0.05 or P < 0.01). The sperm apoptosis rate was positively correlated with the level of ROS in seminal plasma in leukocytospermia group(r=0. 573, P < 0.05). Conclusion The increased sperm apoptosis rate and level of seminal plasma ROS may be related to the infertility of patients with VC and leukocytospermia. The increased level of seminal plasma ROS may be one of the causes of increased sperm apoptosis rate in patients with leukocytospermia.

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

Objective To examine the gene expression levels of matrix metalloproteinase-1(MMP-1), tissue inhibitor of metalloproteinase-1 ( TIMP-1) in peripheral blood mononuclear cells (PBMC) and sera in the patients with chronic hepatitis B (CHB) and to investigate the value of message RNA(mRNA) expression of MMP-1 and TIMP-1 for diagnosing liver fibrosis. Methods PBMC and sera samples were collected from 37 CHB patients and 20 healthy controls. The total RNA isolated from PBMC was reversely transcribed into cDNA. The mRNA levels of MMP-1 and TIMP-1 in PBMC were examined by real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR). The serum levels of MMP-1 and TIMP-1 were determined by sandwich enzyme-linked immunosorbent assay (ELISA). Liver tissues were obtained from all these patients by biopsy and subsequently used for evaluating liver fibrosis stages (S). Intergroup comparison was performed by non parametric test. The correlation analysis was performed by Spearman. Results The MMP-1 and TIMP-1 mRNA levels in PBMC from healthy controls were low. The MMP-1 mRNA levels in PBMC from CHB patients were not significantly different from those in healthy controls,while the TIMP-1 mRNA levels were remarkably higher in CHB patients' PBMC compared to healthy controls. Both the MMP-1 mRNA levels in PBMC and the MMP-1 protein levels in sera were not significantly different among CHB patients at different disease stages and healthy controls (χ~2 =8. 960,P=0.111l ;χ~2 =7. 898, P = 0.211). However, the TIMP-1 mRNA levels in PBMC and the TIMP-1 protein levels in sera increased gradually along with the disease progressed from S1 to S4. The TIMP-1 mRNA levels in PBMC were (1.67±0. 84) lg copy/μL, (3. 48±2. 08) lg copy/μL,(5. 86±3. 47) lgcopy/μL and (8. 14 ± 6. 48) lg copy/μL from stage 1 to 4 respectively, while the protein levels of TIMP-1 in sera were (233. 73±64. 84) ,μg/L, (262. 10±71. 12) μg/L, (301. 15±62. 74)μg/L and(381. 15 ± 152. 75)μg/L, respectively. The differences between each stages were statistically significant (χ~2'= 14. 290, P=0.002,χ~2 = 12.209, P=0. 007). The TIMP-1 mRNA levels in PBMC and the TIMP-1 serum levels were positively correlated with liver fibrosis stage (r=0. 752, P<0. 01;r=0. 530, P=0. 008). Conclusions The TIMP-1 mRNA level in PBMC and TIMP-1 protein level in serum are closely related with liver fibrosis stages. These two parameters, especially the TIMP-1 mRNA level in PBMC, can be potentially new markers for diagnosing liver fibrosis.

ObjectiveTo ascertain the safety and function of the transplantation of neonatal pig islets (NPIs) for diabetic patients.MethodsNPIs were injected into the hepatic artery of 22 patients.After the transplantation,the patients were treated with a multiple drug immunosuppressive regimens.The first 14 patients were treated with cyclosporine (CsA),mycophenolate mofetil (MMF) and prednisolon,and porcine C-peptide was not monitored,the following 2 patients were given cyklosporin and MMF only,while the next 6 patients were given a quadruple drug regimen consisting of OKT3,takrolimus,sirolimus and prednisolon.The blood glucose levels,exogenous insulin requirement,HbA1c,porcine endogenous retrovirus (PERV) and liver function were assessed before and after NPI transplantation.The serum porcine C peptide were monitored in last 8 patients.ResultsThe first 14 patients required less insulin and the HbAlc dropped after the transplantation.In the 2 subsequent patients,the metabolic parameters remained unchanged and monitor of porcine C-peptide was negative.Insulin requirements were reduced in all 6 patients,and HbAlc was normalized 3 months after the transplantation.Significant levels of porcine C-peptide were detected in the patient serum.Two of the patients were given a second injection of NPIs,and one of them became insulin independent for 7 d.No serious adverse events were noted after the transplantation.There was no evidence of PERV transmission.Six out of the 22 patients were followed up for 4-6 years after the NPIs injection,immunosuppressive treatment was stopped 1 year after the transplantation.The patients started to take insulin at the time of follow up.Four patients restricted the intake of sugar,while the other 2 did not.One patient had ketoacidosis twice and slight diabetic retinopathy,and another patient had ketoacidosis induced by acute gastroenteritis.The remaining 4 patients did not have any complications.Assays for PERV were again negative.ConclusionXenogenic islets can survive and function in the human body.No serious adverse events are noted.

Objective To observe the effects of exogenous interleukin (IL)-6 on survival time and regeneration of liver grafts in an orthotopic liver transplantation model in IL-6 knockout (KO) mice.Methods A model of liver transplantation in C57BL/6 wild type (WT) and IL-6 KO mice with C57BL/6 background was established. Grafts were preserved at 4 ℃ in UW solution before transplantation.Study was divided into 4 groups:IL-6 KO→IL-6 KO control group,IL-6 KO→C57BL/6 WT group,IL-6 KO→IL-6 KO+ rIL-6 group,and IL-6 KO→C57BL/6 WT+ rIL-6 group.Recipient animals were injected subcutaneously with human recombinant IL-6 (1 mg/kg body weight) 1 h before transplantation.Survival of liver grafts after transplantation was followed up.Hepatocyte replication with BrdU uptake after liver transplantation was examined by irnmunohistochemistry.Results A total of 48 liver transplantations were performed.Cold ischemic time was 50 min.The survival time of liver grafts in the control group was 2.2 days,and that was 1.9 days,＞17.6 days and ＞20.4 days in IL-6 KO→C57BL/6 WT group,IL-6 KO+ rIL-6 group and C57BL/6 WT+ rIL-6 group respectively with the the difference being statistically significant among the latter three groups (P＜0.01 ).The liver grafts transplanted in control group recipients showed patchy areas of necrosis and hepatocyte ballooning.Slight increase in BrdU uptake was found at 48 h post-transplantation.Minimal injury and no necrosis were observed in IL-6 treated groups.Mild increase of BrdU uptake was demonstrated at 48 h after liver transplantation. Conclusion Exogenous recombinant IL-6 appears to significantly prolong the survival time of IL-6 KO liver grafts after liver transplantation and enhance the regenerative response after liver transplantation in IL-6 KO mice.

Objective To evaluate in vitro effects of specific small interfering RNA (siRNA)-silencing of the casitas B-lineage lymphoma b (Cbl-b)gene on immunocompetence of primary murine lymphocytes. Methods Spleens were resected from C57BL/6 mice, and splenic lymphocytes were sterily isolated and cultured in vitro. These lymphocytes were divided into 3 groups: silence group transfected with a Cbl-b-specific siRNA using the EntransterTM-R 4000 reagent, negative control group transfected with a negative control siRNA using the EntransterTM-R4000 reagent, blank control group receiving no treatment. After additional culture for 72 hours, ELISA was performed to measure levels of interferon γ(IFN-γ)and tumor necrosis factor α (TNF-α)in culture supernatants of lymphocytes. In addition, the Cbl-b gene-silenced lymphocytes were co-cultured with B16F10 melanoma cells to evaluate their immunocytotoxic effects on melanoma cells. Results Splenic lymphocytes were successfully isolated from C57BL/6 mice and cultured in vitro, and the Cbl-b-specific siRNA was also successfully transfected into the primary murine lymphocytes and effectively down-regulated the expression of Cbl-b gene in them. Compared with the negative control group and blank control group, the silence group showed significantly increased supernatant levels of IFN-γ and TNF-α(all P < 0.05). The immunocytotoxic effect of lymphocytes on melanoma cells was significantly stronger in the silence group than in the negative control group. Conclusion Cbl-b gene silencing can promote secretion of IFN-γ and TNF-α by murine lymphocytes, and enhance their immunocytotoxic effects on B16F10 melanoma cells in vitro.

Objective To evaluate the effect ofT-cell immunoglobulin and mucin domain-3 (TIM-3) on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells.Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed.Then,the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supernatants containing TIM-3 and Ig-tail respectively.C57BL/6 mice were immunized with the TRP-2180-188 peptide vaccine for 4 sessions.One week after the last vaccination,C57BL/6 mice were sacrificed,and spleen lymphocytes were collected and then cultured with the TRP-21180-188 peptide and interleukin-2 (IL-2) for 5 days,with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group.Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours (blank control group),TIM-3-containing supernatants (TIM-3 group) and Ig-tail-containing supernatants (negative control group) separately.After 24 and 48 hours of co-culture,cell counting kit-8 (CCK-8) assay was performed to estimate the proliferative activity of lymphocytes,enzyme-linked immunosorbent assay (ELISA) to determine the supernatant levels of interferon (INF)-γ and tumor necrosis factor (TNF)-α,flow cytometry to determine the percentage of CD8 + T cells in the co-culture system.Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid.After 48-hour culture,TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively.As CCK-8 assay showed,the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blank control group and negative control group after 24-and 48-hour culture (78.06％ ± 6.37％ vs.100.00％ ± 10.42％ and 108.70％ ± 9.90％ at 24 hours,42.93％ ± 5.93％ vs.100.00％ ± 6.24％ and 168.00％ ± 2.98％at 48 hours,all P ＜ 0.05),so was the ratio of cellular proliferative activity at 48 hours to that at 24 hours (all P ＜ 0.05).Compared with the blank control group and negative control group,the TIM-3 group showed significantly decreased supernatant levels of IFN-γ and TNF-α after 24-hour (IFN-γ:192.96 γ 5.05 ng/L vs.216.44 ± 7.85 ng/L and 223.67 ±7.79 ng/L,both P＜ 0.05;TNF-α:58.43 ± 0.26 ng/L vs.26.43 ± 0.01 ng/L and 86.85 ± 1.12 ng/L,both P＜ 0.05) and 48-hour culture (IFN-γ:54.95 ± 0.57 ng/L vs.230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L,both P ＜ 0.05;TNF-α:30.23 ±0.26 ng/L vs.26.84 ± 0.20 ng/L and 45.34 ± 0.22 ng/L,both P ＜ 0.05).In addition,the median percentage of CD8+ T cells was significantly increased in the TIM-3 group compared with the blank control group and negative control group after 24-and 48-hour culture (3.30％ vs.0.421％ and 2.22％ at 24 hours,4.06％ vs.0.577％ and 0.691％ at 48 hours,all P＜ 0.05).Conclusion TIM-3 in vitro can suppress the proliferative activity of and secretion of IFN-γand TNF-α by lymphocytes,but increase the percentage of CD8 + T cells in the co-culture system of TRP-2180-188 peptide-stimulated lymphocytes and B16F10 cells.

Objective To investigate the characteristic of P30o event-related potentials(ERP) of impulsive behavior in heroin addicts.Methods The Iowa gambling task (IGT) were performed by using the paradigm for psychological experiment both in heroin addiction group (HA group) and health control group (HC group),the inspection of electroencephalography were underwent in all the subjects concurrently.Following the collection of data of ERP,amplitude and latency of P300 were compared between the two groups,and then the relationship betweenamplitude or latency of P300 and the results of Barratt impulsiveness scale were analyzed separately.Results Compared to HC group,BIS score as well as the numbers of high frequency loss cards were significantly higher in HA group ( HA:75.12 ± 12.49,91.14 ± 21.35 ; HC:66.54 ± 8.61,73.71 ± 18.91 ; P ＜ 0.05 ),while the both two groups had visible waveforms of P30o,and the amplitude and latency were markedly lower ( HA:4.92 ± 1.14,293.43 ± 36.21 ; HC:7.65 ± 1.59,332.68 ± 40.15 ; P ＜ 0.05 ) and were negatively associated with BIS score in HA group( r =-0.76,-0.52,P＜ 0.05).Logistic regression results showed that the scores of BIS-11 were related to amplitude of P30o merely( P ＜ 0.05 ).Conclusion Impulsive behavior can be observed from the abnormal characteristic of the P300 event-related potential of impulsive behavior in heroin addicts,which may partly contribute to both addiction and relapse of heroin addict.