Objective To evaluate the effects of propofol and thiopontal on calcium and potassium channels in rat ventricular myocytes and to elucidate the underlying mechanisms of their inhibitory effect on myocardium. Methods Freshly isolated ventricular myocytes were prepared from hearts of rats by trypsin. The effects of propofol and thiopental on L-type calcium current(Ica) and delayed rectifier potassium current(IK)were compared using whole-cell patch clump technique. Results Propofol and thiopontal produced a concentration-dependent inhibition of Ica. Peak concentration of propofol(50 ?mol?L~(-1)) and thiopental(100 ?mol?L~(-1)) during induction of anesthesia decreased Ica by 28% and 46% and shifted the steady-state inactivation curve to more negative voltage, but had no effect on the steady-state activation curve. Propofol and thiopental also decreased IK in a concentration-dependent manner, but the effects of both anesthetics on IK were smaller compared with their effects on Ica. Conclusion The findings of this study suggest that the negative inotropic of propofol and thiopental are, at least in part, related to decrease in Ca~(2+) trans-sarcolemmal current by accelerating L-type calcium channel inactivation. Both anesthetics decrease delayed rectifier potassium current, thus partially antagonizing the effect of decreased calcium current.

AIM: To observe the effect of oxymatrine on sodium channel in isolated cardiomyocytes of guinea pig. METHODS: Single ventricular myocytes of guinea pigs were obtained by enzymatic dissociation. The whole - cell patch clamp recording technique was used to record the change of sodium current by different dosage of oxymatrine from 1 to 1 000 ?mol?L-1 . RESULTS: Oxymatrine decreased sodium current in a dose - dependent manner. Oxymatrine (100 ?mol?L-1) decreased the current density by 40% (P

Ojective To investigate the efficacy and complication of percutaneous transhepatic plastic stent implantation for maligant obstructive jaundice. Methods Percutaneous transhepatic plastic stent implantation was performed in 32 patients with malignant obstructive jaundice. The early and long term theraputic efficacy and complications of the technique in 32 patients were observed for follow-up periods of 35～376 days. Results Technical success rate was 100%. Serum total bilirubin decreased significantly the first week after percutaneous transhepatic plastic stent implantation ( t =3.643, P

Objective To investigate comparability between Sysmex different hematology analyzers.The consistency of all he-matology analyzers with the same fresh sample.Methods Selected Sysmex XE-2100 as reference instrument which attend external quality assessment.Fresh high,medium and low blood were detected in Sysmex XS-800i,Sysmex XT-4000i and Sys-mex XT-1800i,compared results of WBC,RBC,HGB,HCT,MCV and PLT.Results The bias of XS-800i and XE-2100 was WBC 2.85%,RBC 1.44%,HGB 0.75%,HCT 2.11% and PLT 5.53%.The bias of XT-4000i and XE-2100 was WBC 1.26%,RBC 0.95%,HGB 0.68%,HCT 1.35% and PLT 2.68%.The bias of XT-1800i and XE-2100 was WBC 5.21%, RBC 1.96%,HGB 1.60%,HCT 1.96% and PLT 4.95%.It had good compatability.All various parameters was in the al-lowed range.Conclusion Should maintenance and compare the hematology analyzers periodically,found the problem then calibration instruments in time,ensure the consistency of measurement results between different instruments and guarantee accuracy of the results.

Objective To probe the biological effect of multiple intraepithelial injections of recombinant adenovirus-p53(rAd-p53)on inducing the apoptosis in patients with dysplastic oral leukoplakia.Methods 18 patients clinically and histopathologically diagnosed as dysplastic oral leukoplakia were recruited for this study.Intraepithelial injections of rAd-p53 were administered.Immunohistochemistry was used to examine the protein expression of p53 and bcl-2.TUNEL was performed to detect apoptotic cells.Results In the post-treatment patients,p53 protein expression were significantly enhanced(100 ％,18/18),yet bcl-2 protein presented low expression(17 ％,3/18).Statistical analysis revealed the expression of p53 protein had a negative correlation with that of bcl-2 protein(r =-0.837,P ＜ 0.01).15 post-treatment samples (83 ％)were detected obvious apoptotic cells,especially in the samples that were strong p53-positive(r =0.684,P ＜ 0.01).Conclusion Intraepithelial injections of rAd-p53 can induce apoptosis for patients with dysplastic oral leukoplakia.It may be a promising treatment for oral leukoplakia.

Objective To investigate the effects of dihydrofolate reductase (DHFR) gene expression up-regulating on cisplatin resistance in epithelial ovarian cancer cell lines.Methods The cDNA length of DHFR gene was amplified by PCR and was connected to lentiviral vector pWPI, the recombinant retroviral vector DHFR-pWPI was infected SKOV3 cells by lipofectamine 2000.The groups included DHFR-pWPI-SKOV3 cell, pWPI-SKOV3 cell and SKOV3 cell group.Western blot was used to detect the expression of DHFR.Flow cytometry was applied to measure the cell apoptosis rate of 3 groups cells in different cisplatin concentrations (2.5, 5.0, 10.0, 20.0 μg/ml) and at different time period (24, 48 and 72 hours), and half maximal inhibitory concentration (IC50) treated with cisplatin concentration (6.0, 4.0, 4.9 μg/ml).High performance liquid chromatography (HPLC) was applied to test intracellular cisplatin concentration in different cisplatin concentration (4.0, 6.0, 8.0 μg/ml) at 24 and 48 hours.Transmission electron microscope was used to observe ultrastructural changes cells under IC50 cisplatin concentration.Results The recombinant plasmid DHFR-pWPI was constructed and then infected into SKOV3 cell successfully.(1) The expression of DHFR detected by western blot in transfection group was higher than those in the negative control group and blank control group (10.280±0.009 vs 2.050±0.003 vs 3.480±0.003;P＜0.01).(2) Treated with cisplatin concentration (2.5, 5.0, 10.0, 20.0 μg/ml) at 24, 48 hours, the apoptosis rate detected by flow cytometry results shown that they were lower than those in the negative control group and blank control group (P＜0.05), while treated at the concentration of 5.0 and 10.0 μg/ml for 72 hours, whose apoptosis rate in transfection group was higher than those in the negative control group and blank control group (P＜0.05).When treated cells under IC50 cisplatin concentration (6.0, 4.0, 4.9 μg/ml) at for 24 and 48 hours, the results indicated thatthere were mainly G0/G1 stage cell cycle rate in 3 groups, it was obviously higher in transfection group than those in two control groups (P＜0.05).However, mainly G2/M, S stage cell cycle rate for 72 hours, and S stage cell cycle rate in transfection group obviously higher than those in two control groups, but G2/M stage cell cycle rate were lower (P＜0.01).(3) After treated with cisplatin concentration (4.0 μg/ml) for 24, 48 hours and cisplatin concentration (6.0 μg/ml) for 24 hours, the intracellular cisplatin content tested by HPLC method in the transfection group were significantly lower than those in two control groups (P＜0.01).While, at 6.0 μg/ml of cisplatin concentration for 48 hours and 8.0 μg/ml of cisplatin concentration for 24 and 48 hours, the intracellular cisplatin content of transfection group were obviously higher than those two control groups (P＜0.05).(4) Treated with IC50 (6.0, 4.0, 4.9 μg/ml) cisplatin concentration at different time to obeserve ultrastructural changes by transmission electron microscopy.The results shown that the microwire gathered together at 24 and 48 hours, and the number and structure of mitochondria had obvious change in transfection group, while there was rare microfilament, the number of mitochondria decreased but structure change was not apparent in two control groups.There were appeared expansion of endoplasmic reticulum and had rare normal organelles among three groups.After treated with cisplatin for 72 hours, there were inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in two control groups, and there were rare microfilament in transfection group, nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells on the verge of death.Conclusion The lentiviral expressing vector harboring human DHFR gene were successfully constructed.When the up-expression of DHFR gene, the drug-resistant in ovarian cancer cell may be increase, which suggest that there were certain contact between resistance increases with microfilament gathered and the change of the mitochondria.

Background and purpose:Dihydrofolate reductase (DHFR) is expressed highly in platinum-resis-tant ovarian cancer. This study aimed to explore the relationship between the silence ofDHFR gene and platinum drug resistance in ovarian cancer, and lay the foundation for the treatment of platinum-resistant ovarian cancer.Methods:To design targeting hairpin siRNA ofDHFR gene, the optimal siRNA silent sequence was selected, and lentiviral vector carryingDHFR gene was constructed successfully, named DHFR-pGCSIL-SKOV3 cell. Flow cytometry was used to detect the cell apoptosis of DHFR-pGCSIL-SKOV3 cells, pGCSIL-SKOV3 cells and SKOV3 cells incubated in various concentrations of cisplatin (2.5, 5.0, 10.0 and 20.0 μg/mL) at different time points (24, 48 and 72 h), and cell cycle changes of these cells at IC50 cisplatin concentration (4.4 μg/mL). High performance liquid chromatography was used to test intracellular concentration of cisplatin at different induction concentration of cisplatin (2.5, 5.0 and 7.5 μg/mL) and various time points (24 and 48 h). Ultrastructural changes of these cells at concentration of cisplatin IC50 (4.4 μg/mL) were observed by transmission electron microscope.Results:After annealing double-strand nucleotide was connected to pGCSIL/GFP vector, sequencing result was correct. SKOV3 cell were transfected with virus particles followed by Western blot detection of interference effect. Flow cytometry was used to detect apoptosis in three groups of cells, and increased apoptosis rate was found at the raised cisplatin concentration (2.5, 5.0, 10.0 and 20.0 μg/mL) at 24, 48 and 72 h in DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3 and SKOV3 cells. The apoptosis rate in DHFR-pGCSIL-SKOV3 was signiifcantly higher than that in pGCSIL-SKOV3 and SKOV3 cells at 24 and 48 h (P<0.05). Flow cytometry was adopted to test cells cycle of 3 groups at different time period under IC50 cisplatin concentration (4.4 μg/mL), the results indicated that G0/G1 phase cell rate of DHFR-pGCSIL-SKOV3 was much more than the others, of which G2/M and S phase cell rates were on the contrary. While at 72 h, 3 groups were mainly G2/M and S phase cell rates, DHFR-pGC-SIL-SKOV3 was lower than the others. High performance liquid chromatography method was used to detect intracellu-lar cisplatin concentration at 24 and 48 h after the cells were incubated at various concentrations of cisplatin (2.5 and 5.0μg/mL). The results showed the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was signiifcantly higher than that of pGCSIL-SKOV3 and SKOV3 cells. However, after incubation at cisplatin concentration of 7.5 μg/mL, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was signiifcantly lower than that of pGCSIL-SKOV3 and SKOV3 cells at 24 h, while higher than pGCSIL-SKOV3 and SKOV3 cells at 48 h (P=0.034,P=0.014). We observed ultrastructural changes of three different cell lines induced by IC50 cisplatin concentration(4.4 μg/mL) at different time points by the electron microscope. We found that the microiflaments were increased and gathered together and mitochondrial structure was also changed obviously without the drug. However, there was rare microiflament in three groups of cells at 24 and 48 h, while at 72 h, obviously increased inordinate microiflaments were observed.Conclusion:We successfully constructed pGCSIL lentivirus interference carrier carryingDHFR gene. The research indicates that down-regulation ofDHFR gene is related to cisplatin drug resistance in ovarian cancer. The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.

Purpose To investigate the effects of different concentration of ketamine on Ca2 transsarcolemmalinflux induced by KCl in isolated rat ventricular myocytes. Methods Freshly isolated rat ventricular nyoeyteswere loaded with Fluo-3AM, a Ca2 + indicator. The effects of different concentration ketamine( 1 × 10- s, 1 × 10- 4,1 × 10-3 mmol/L) on the change of intracellular Ca2+ concentration induced by KCl were investigated. ResultsLow concentration ketamine(1 × 10-5 mrnol/L) did not change Ca2+ transsarcolemmal influx. Although mediumeoncentration ketamine( 1 × 10-4 rmol/L) made the influx slower, the eventual peak concentration of intracellularCa2+ had no difference from that of the control group. The high concentration ketamine (1 × 10-3 mmol/L) inhibited Ca2-1 influx,intracellular Ca2+ fluorescent intensity decreased about 13.2% (P＜0.05). ConclusionsKetamine inhibits Ca2 + trranssarcolemmal influx in isolated rat ventricular myocytes dosedependently, which may inpart explain its negative inotropic effect.

Objective Stress effect plays an important role in the development of some myocardial diseases. We hypothesized it was important nosogenesis to myocardial damage and cardiac allograft vasculopathy. Methods The transplanted hearts from Lewis to Wister rats served as allografts and from Lewis to Lewis rats as isografts based Ono's model. The differential proteins in the transplanted hearts were separated by comparative proteome, and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and searched by Matrix Science software system.Results All transplanted hearts were characterized by lumen loss [(total vessel area-luminal area)/total vessel area] in the coronary artery 2 weeks after the operation [(2.07%±0.93%) vs. (27.58%±11.14%), P＜0.01], but more predominant after 8 weeks [(2.34%±1.06%) vs. (72.29%±20.57%), P＜0.01]. All samples of the left ventricle were analyzed by proteomic techniques and 37 distinct proteins involving their respective isoforms and subunits were identified. Nine proteins were correlated to endoplasmic reticulum stress effect and myocardial damage, and 2 proteins were verified by Western blot.Conclusion Stress plays an important role in cardiac allograft damage and the development of rat cardiac allograft vasculopathy.

OBJECTIVE To explore the absorption mechanism of thiophenorphine, and its effect on P-glycoprotein (P-gp) expression by using Caco-2 cell monolayer model. METHODSThe LC-MS-MS method was applied to determine thiophenorphine concentration in millicell system. The bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption. P-gp inhibition was studied by flow cytometry using calcein-AM as P-gp substrate.The expression of P-gp was evaluated using Western blotting. RESULTSThiophenorphine transport in Caco-2 cells was in time-dependent manner. Its average apparent permeability coefficient (P_(app)) was 2.338×10~(-6) cm·s~(-1). P_(app) was increased 2.8 folds by P-gp inhibitor ciclosporin A, and 2.3 folds by mulitdrug resistance-associated protein2 (MRP2) inhibitor MK571. The accumulation of calcein-AM and the expression of P-gp in Caco-2 cell line wasn't changed noticeably by thiophenorphine. CONCLUSION Thiophenorphine is a common substrate of P-gp and MRP2 and it shows normal transport in millicell system. The expression of P-gp doesn't induce by thiophenorphine.