Effects Of Morphine And Low-dose Ketamine On T Cells Of Patients With Refractory Cancer Pain In Vitro

[Object]Refractory cancer pain is very severe,and patients' quality of life is disturbed severely.Study has proved that PCA of morphine and ketamine for analgesia was safe and efficacious to many patients.PCA can maintain serum drug level constancy and efficiency,and stable analgesia effect can be obtained.The opioid of first choice for moderate to severe cancer pain is morphine.In a small minority of patients,adequate relief without excessive adverse effects may depend on the use of alternative opioids,spinal administration of analgesics or non-drug methods of pain control.And ketamine may have exerted an analgesic effect by attenuating central sensitization and opioid tolerance.But all of refractory cancer pain,morphine and ketamine have been reported to produce a number of immunomodulatory effects.The long-term and intensive refractory cancer pain would lead to cacoethic immune suppression,and the suppression would aggravate the state of immune suppression caused by tumour progression and chemoradiotherapy.Chronic administration of morphine affects both innate and adaptive immunity.Immunosuppression of ketamine is the lowest among the general anesthetic.Subthreshold dose ketamine has little effect on immunocyte and susceptivity of tumor metastasis,and in clinic we often use subthreshold dose to relieve effect of ketamine on immune function.But there is less report about the effect of morphine in combination with ketamine on T cells of patients with refractory pain.The current study was performed to assess the direct effect of morphine and low-dose ketamine on T cells of patients with refractory cancer pain in vitro.[Method]All patients were finally diagnosed as malignant tumour,and were received oral analgetica of three-step analgesic ladder advocated by WHO,but received less effect.The criteria of exclusion:cases diagnosed as tumour with concurrent infection,ulceration with pain;cases being dead in one month or the therapeutic regimen being changed;cases with dyscrasia,infection,extensively metastased to distant organ;cases with damaged function of liver,heart and lung.And informed,written consent was obtained from the final 46 patients included.Peripheral blood mononuclear cells(PBMCs)were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient,and counting,activity determination of PBMCs was then done.T cells were isolated by positive selection using anti-CD3 beads,and confirmed by fluorescence-associated cell sorting(85%purity).T cells were adjusted to a final concentration of 1×105 cells/ml in 96-well plate.T cells were cultivated at 37? and 5%CO2 in RPMI 1640 medium supplemented with 10%fetal calf serum and antibiotics.T cells were then treated with vehicle,morphine(200 ng/ml),or morphine(200ng/ml)and ketamine(100 ng/ml)for 24 h before stimulation with anti-CD3 and anti-CD28(5ug/ml),and then stimulation lasted for another 24h.Then flow cytometric analysis of trichromatism monoclonal antibody directly labelling was used to detect CD4+CD3+ T cell and CD8+CD3+ T cell,and then the ratio of CD4+CD3+ T cell and CD8+CD3+ T cell was gotten,from that we could determine the change of T cell population and the ratio of subgroup.Supernatant IL-2 and IFN-y immunoreactive protein concentration was measured by cytokine-specific enzyme-linked immunosorbent assay kits.IL-2 mRNA and IFN-? mRNA was detected by quantitative real-time RT-PCR,to determine the change of IL-2 and IFN-y in transcriptional level.Activated P65 NF-?B was detected by western immunoblot analysis,to investigate whether the effect of morphine and ketamine on IL-2 mRNA and IFN-y mRNA transcription is the result of a decrease in DNA protein binding to cognate DNA elements.Statistical comparisons between experimental groups were performed where appropriate with SPSS 17.0.All data are expressed as M±S.Statistical significance was analyzed using one-way analysis of variance(ANOVA),and chisquare test was ued for enumeration data.Difference was considered significant if P<0.05,and difference was considered obviously significant if P<0.01.[Result]Compared with the control group,both CD4+ and CD8+ T cells were decreased by morphine,and they were also decreased by morphine and ketamine(P<0.05).And CD4+/CD8+ T cells ratio in morphine group and morphine+ketamine group both decreased,compared with that in the control group(P<0.05).However,there was no significiant difference of all of CD4+,CD8+ T cells and CD4+/CD8+ ratio between morphine group and morphine+ketamine group.There was no significiant difference of supernatant IL-2 and IFN-y among the control group,the morphine group and the morphine+ketamine group.However,IL-2 mRNA and IFN-? mRNA in morphine group and morphine+ketamine group both decreased,compared with that in the control group(P<0.05).And there was no significiant difference of IL-2 mRNA and IFN-? mRNA between morphine group and morphine+ketamine group.In western immunoblot analysis,activated P65 NF-?B in morphine group and morphine+ketamine group both decreased,compared with that in the control group(P<0.05).And there was no significiant difference of activated P65 NF-?B between morphine group and morphine+ketamine group.[Conclusion]In this study,in vitro,we observed that morphine in combination with low-dose ketamine acted on T cells from peripheral blood of patients with refractory cancer pain,and determined change of T cell subpopulation,production of cell factor IL-2,IFN-y and their mRNA,and nuclear factor NF-?B,by the experiment technology of flow cytometry,ELISA,PCR and Western,et al.From the observation,we confirmed the conclusions as follows:1.Both morphine and morphine in combnition with low-dose ketamine suppressed some immune function of actived T cells of patients with refractory cancer pain in vitro.2.The extent of morphine in combnition with low-dose ketamine suppressing immune function of actived T cells was the same with the extent of morphine alone.3.The nuclear factor NF-?B may participate in the process of morphine and ketamine suppressing immune function of actived T cells.From this study of the preliminary observation on effect of morphine in combination with low-dose ketamine on T cells from peripheral blood of patients with refractory cancer pain,experimental basis was offered for application of the treatment,but more study should performed on more immunocyte and the effect in vivo.