Effects Of Fentanil Remifentanil And Sufentanil On The Immune Function Of Dendritic Cells From Umbilical Cord Blood

Objective: The purpose of the present study is to evaluate the effects of fentanil remifentanil and sufentanil (the analgesia and anaesthesia concentration) on the immune function of dendritic cells from umbilical cord blood.Methods: Normal fetal umbilical cord blood (about 70ml) of healthy parturient women was collected under sterile condition, and anticoagulated with heparin. At the superclean bench the cord blood was diluted with saline according to the volume of 1:1 and then the diluted blood was slowly added into the upper stratum of lymphocyte isolation. Cord blood mononuclear cytes (CBMC) were separated by centrifugal in density gradient. The supernatant was removed after the cells were washed with saline three times. Then the cells were numbered adjusting its number to 2×106/ml with serum-free medium (SFM) and then inoculating into a 24-pore culture plate. There was 2ml in each pore. The suspended cells were discarded after cultured for 2 hours in the incubator (37℃,5%CO2 ) and the attached cells were adjusted to1.0×105 /ml with SFM. The cells were randomly divided into four groups: The normal control group (group N), and the drug group (group F, group R, group S). Group N was added by rhGM-CSF, rhIL-4 while incubated with rhTNF-α(50ng/ml) for 14 days in order to obtain the matured DCs. Group S was added by rhGM-CSF, rhIL-4, rhTNF-αat the same time incubated with fentanil 1, 5, ng/ml (F₁,F5) respectively for 14 days. Group R was added by rhGM-CSF, rhIL-4, rhTNF-αat the same time incubated with remifentanil 1, 5ng/ml (R₁,R5) respectively for 14 days. Group S was added by rhGM-CSF, rhIL-4, rhTNF-αat the same time incubated with sufentanil 0.1, 0.5 ng/ml (S_0.1,S0.5) respectively for 14 days. The growth and morphology of DCs were identified by inverted optical microscope. The phenotypes of 14-day-cultured DCs (CD80 , CD86 ,CD83,HLA–DR) were identified by flow cytometry. IL-12 level in supernatant was determined by enzyme linked immunosorbent (ELISA) assay. The peripheral blood (about 15 ml) was taken from the volunteers and peripheral blood mononuclear cells (PBMC) were separated. The PBMC were cultured for 2 hours in the incubator and then suspension cells were collected to prepare allogeneic mixed leukomonocytes. The suspension cells was adjusted to 2.0×10⁶ /ml and cultured for seven days as the reactive cells. The cells cultured for ten days in different groups were collected as the stimulant cells. The ability of DCs to stimulate the proliferation of the allogeneic mixed lymphocyte was detected with methyl thiazolyl terazolium (MTT) assay.Results:1 Effects of fentanyl, remifentanil and sufentanil on the growth and morphology of DCs: In each group there were more attached cells aggregating to cluster and few suspended cells after cultured for 24 hours in the incubator, After the control cells cultured 5d significant colony-forming drug group remifentanil 5ng/ml (R₅) group sets the number of colony-forming small and less; cultured cell volume increased after 14d shows a typical dendritic-like processes, while the Ruifen too Nepal 5ng/ml (R₅) group of cells, dendritic pseudopodium was not obvious, and there is a typical dendritic-like changes in the number of small cells ( Fig.1 4).2 Effects of fentanyl, remifentanil and sufentanil on the expression of CD80CD86: Levels of CD80, CD86, CD83 and HLA-DR molecule expression in the control group and the F₁ (fentanyl 1ng/ml) group showed no significant difference (P>0.05); R₁, S_0.1, with the F₁ team to compare their expression was significantly lower (P<0.05), but the R₁ group, S_0.1 group no significant difference between the two groups (P>0.05); R5 group and F5 group, S_0.5 group to compare their expression was significantly lower (P<0.01), F₅ group, S_0.5 group difference was not statistically significant (P>0.05).3 Effects of fentanyl, remifentanil and sufentanil on the level of the IL–12 production: the control group and the F₁ group showed no significant difference (P>0.05); F₁ group, S_0.1 group, R₁ group between the two groups (P<0.05) a statistically significant difference, R₁ group DCs secrete cytokines IL-12 inhibition of the most obvious; F₅ group, S_0.5 group, R₅ group between the two groups (P<0.05) a statistically significant difference, R₅ Group DCs secrete cytokines IL-12 inhibition of the most obvious; R₅ compared with the above-mentioned IL-12 secretion volume reduction (P<0.05), compared with control group (P<0.01). The results were: 920.07±112.89,818.18±74.00,564.30±22.58,661.01±38.20,385.05±82.59,427.38±71.01,214.54±22.40.4 Effects of DCs stimulated by fentanyl, remifentanil, and sufentanil on the proliferation of the allogeneic mixed lymphotyte: mature DCs on the proliferation of mixed lymphocyte has a strong stimulating effect, while the immature of DCs to stimulate T cell proliferation weak, drug group and control group cells can promote the same kinds of mixed lymphocyte proliferation in the control group, but the ability to stimulate stronger than the medicine group (P <0.05), and with the ability to stimulate the gradual increase in the concentration of drugs decline.Conclusion: The umbilical cord blood mononuclear cells of origin in fentanyl, remifentanil, or under the influence of sufentanil induction of mature dendritic cells, analgesic concentration of fentanyl on the mature dendritic cells and immune function had no significant effect (1ng/ml), inhibitory concentration of remifentanil incision reaction the greatest influence (5ng/ml). Other concentrations of fentanyl, sufentanil, remifentanil on dendritic cells have a certain influence, but no difference between the two groups.