Objective To evaluate the relationship between protein kinase B (AKT) and protein kinase (PKCθ) and morphine-induced inhibition of differentiation of T helper (Th) cells.Methods Peripheral venous blood samples were taken from 20 healthy volunteers..Peripheral blood mononuclear cells (PBMCs) were extracted by density gradient centrifugation method.CD4+ T lymphocytes extracted were purified by magnetic bead separation.CD4+ T lymphocytes were randomly assigned into 5 groups (n =3 each) using a random number table.CD4 + T lymphocytes were incubated routinely in group C.CD4 + T lymphocytes were incubated in the presence of PMA 25 ng/ml + ionomycin 1 μg/ml (group PI),PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μμg/ml (group M),or PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μg/ml + naloxone 50 μμg/ml (group N).The cells were incubated for 4 h in the incubator containing 5％ CO2 at 37 ℃.The expression of interferon (IFN)-γ and interleukin-4 (IL-4) was detected by flow cytometry.The expression of IFN-γ and IL-4 was used to reflect the percentage of Th1 and Th2 cells,respectively.The ratio of Th1/Th2 was calculated.The expression of AKT,phosphorylated AKT (p-AKT),PKCθ and phosphorylated PKCθ (p-PKCθ) was detected by Western blot,and the ratio of p-AKT/AKT and p-PKCθ/PKCθ was calculated to reflect the activities of AKT and PKCθ,respectively.Results Compared with group C,the percentage of Th1 and Th2 cells and ratio of Th1/Th2 were significantly increased in PI,M and N groups,the activities of AKT and PKCθ were increased in PI and N groups,and the activity of PKCθ was increased in group M (P ＜ 0.05).Compared with group PI,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly decreased in group M,the ratio of Th1/Th2 was decreased in group N (P ＜0.05),and no significant change in the activity of PKCθ was found in M and N groups (P ＞ 0.05).Compared with group M,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly increased (P ＜ 0.05),while no significant change in the activity of PKCθ was observed in group N (P ＞ 0.05).Conclusion The mechanism by which morphine inhibits the differentiation of Th cells through activating opioid receptors is related to inhibition of AKT activation,but not related to PKCθ.

Objective To evaluate the relationship between GATA-3 and T-bet and inhibition of the differentiation of human T helper cells by morphine.Methods Ten healthy volunteers,aged 20-50 yr,weighing 50-70 kg,were enrolled in the study.Peripheral venous blood samples were taken in the early morning.Peripheral blood mononuclear cells (PBMCs) were isolated and assigned into 5 groups (n=10 each).PBMCs were incubated routinely (group C).PBMCs were incubated in the presence of PMA and ionomycin (group P),morphine 50 μg/ml (group M),morphine 50 μg/ml + naloxone 50 μg/ml (group MN) or naloxone 50μg/ml (group N),and were then stimulated with PMA and ionomycin for another 4 h.The percentage of Th1 and Th2 cells was detected by flow cytometry.The Th1/Th2 ratio was calculated.Interferon (IFN)-γ and interleukin (IL)-4 concentrations in the supematant were determined using ELISA.The activities of GATA-3 and T-bet were analyzed by EMSA.Results Compared with group P,the percentage of Th1 and Th2 cells,Th1/Th2 ratio,IFN-γand IL-4 concentrations in the supernatant,and GATA-3 and T-bet activities were significantly decreased in group M,the percentage of Th1 cells,Th1/Th2 ratio,and IFN-γ concentration in the supernatant were significantly decreased in group MN (P ＜ 0.05 or 0.01).Compared with group M,the percentage of Th1 and Th2 cells,Th1/Th2 ratio,IFN-γ and IL-4 concentrations in the supernatant,and GATA-3 and T-bet activities were significantly increased in group MN (P ＜ 0.05 or 0.01).There was no significant difference in the indexes mentioned above between groups N and C (P ＞ 0.05).Conclusion Morphine inhibits the differentiation of human T helper cells by activating opioid receptors,which may be related to the inhibition of GATA-3 and T-bet activities.

AIM　To study the electrochemical behavior of ofloxacin at Pt/GC ion implantation modified electrode. METHODS　With Pt/GC ion implantation modified electrode as working electrode, the behavior of ofloxacin was studied by voltammetry in 0.40 mol*L-1 KCl solution. RESULTS　A sensitive reductive peak of ofloxacin was obtained by linear sweep voltammetry. The peak potential was -1.35 V (vs SCE). The peak current was proportional to the concentration of ofloxacin over the range of 1.0×10-6-3.0×10-5 mol*L-1 with the detection limit of 5.0×10-7 mol*L-1. The behavior of reduction wave was studied and applied to determination of ofloxacin in tablets. CONCLUSION　The reduction process was irreversible. The element composition, atomicity form and depth of distribution at the surface of Pt/GC electrode were determined by Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS) and scannig electron microscope (SEM). The catalysis behavior and reaction mechanism at Pt/GC modified electrode was also studied.