| Objectives This study was designed to observe the changes of T helper(Th) cells in the colorectal cancer(CRC) patients and to assess the effects of morphine and ketamine on the differentiation of the Th cells in vitro.Methods 20 healthy volunteers(male 10, female 10) and 20 CRC patients(male 10, female 10) ranging in age from 45 to 65 years and BMI from 18 to 25 kg/m2 were enrolled in this study. The peripheral bloods from healthy volunteer(10 milliliters) and CRC patients(20 milliliters) were collected in the morning from fasted subjects. The peripheral blood mononuclear cells(PBMCs) were isolated using density Gradient sedimentation over Histopaque 1077 in cell separation medium. Afterwards, a trypan blue dye test to assure cell viability >95% was performed and then cells were suspended(3×106 cells) in RPMI Medium1640 supplemented with10% calf serum and incubated for 0.5h for later use. Cells isolated from normal subjects were divided into two groups(group P1, C1): control groups without PMA or ionomycin; PMA and ionomycin without ketamine or morphine. Cells isolated from CRC patients were divided into six groups(group P2, C2, K1, K2, K3, M): control groups without PMA+ionomycin; PMA+ionomycin without ketamine or morphine; ketamine(25u M) and PMA+ionomycin; ketamine(50u M) and PMA+ionomycin; ketamine(100u M) and PMA + ionomycin; morphine(50ng/m L) and PMA+ionomycin. According to the above treatments in groups, cells were incubated for 4h at 37? in a 5% CO2 atmosphere. Th subsets were analyzed by FACS Calibur flow cytometer at 4 h later. Th1 cells were labeled as CD3+CD8-IFN-?+ and Th2 cells were labeled as CD3+CD8-IL-4+. The cell counts were presented as percentage to total CD3 positive cells.Results In normal subjects groups, the cells of Th1 and Th2 were significantly increased after the stimulation of PMA and ionomycin in group C1 compared to group P1(p<0.001, respectively). In CRC groups, the cells of Th1 and Th2 were significantly increased after the stimulation of PMA and ionomycin in group C2 compared to group P2(p<0.001, respectively). In the presence of PMA and ionomycin, Th1 cells and Th1/Th2 ratio were significantly decreased in the group C2 compared to the group C1(p<0.001). Compared to the group C2, morphine decreased the number of Th1 cells and Th1/Th2 ratio(p<0.001, respectively), but had not decrease the number of Th2 cells(p=0.37). Compared to the group C2, ketamine did not significantly change Th1 cells, Th2 cells and Th1/Th2 ratio in group K1(p=0.57, 0.71, 0.81 respectively), but significantly increased Th1/Th2 ratio in group K2(p<0.01), and significantly decreased Th1 cells and Th2 cells and increased Th1/Th2 ratio in group K3(p<0.01, respectively). With the increased concentrations, ketamine increased the Th1/Th2 ratio(from1.64 25 u M to 1.71 50 u M, p<0.01; from 1.71 50 u M to 2.03, p<0.01 100 u M).Conclusions CRC shifts the balance of Th1/Th2 towards Th2 by inducing an immunological response. Morphine and ketamine with a higher than subanesthestic concentration can suppress the differentiation of Th cells, but morphine decreases Th1/Th2 ratio and ketamine increases Th1/Th2 ratio. Ketamine can affect the differentiation of Th cells in a dose-dependent model. In clinical practice, the combination of morphine with small-dose ketamine may reduce morphine consumption and the risk of adverse drug reactions, alleviate immune inhibition and improve the quality of life of CRC patients. |
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