Effects Of Sevoflurane On The Growth Of Immature Dendritic Cells And The Vatilis Of Mature Dendritic Cells

Objective: Sevoflurane is a new halogen anesthetics.It's physical and chemical properties are stable. It has many merits ,such as induce rapidly, no irritation to respiratory, inhibition of circulating lightly, wake up fast and completely and no tissue toxicity.Now it has been of a broader used inhaled anesthetics in clinic .The dendritic cells(DCs) are the most strongest antigen–presenting cells which are the only cells that can stimulate the naive T lymphocytes.So DCs have an important role in initiate immune response.Athough the precursor cell of dendritic cells(DCs) experience three stage before presentting antigens ,DCs can differentiate into tow immunophenotypes (immature type and mature type).The purpose of the essay is to study the effects of sevoflurane on the growth of immature dendritic cells and the vatilis of mature dendritic cells from umbilical cord blood,through expousure to sevoflurane.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 and the ratio is 2:1. 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 resuspended and numbered and the density was adjusted to 2×106/ml with serum-free medium (SFM) and then inoculating into a 6-pore culture plate ,with 3ml in each pore. The suspended cells were discarded after 2 hours incubation, (37℃,5%CO2, 95%air) and the attached cells were adjusted to 1×106 /ml with SFM. The cells were randomly allocated to three groups: The normal cultured group (group C), the immature type group (group I) and the mature type group (groupM). Group C was cultured in the presence of rhGM-CSF (50ng/ml) and rhIL-4 (10ng/ml) for 14 days and rhTNF-α(50ng/ml) was added into till the 12th day. Cells of Group I and Group M were exposured to 2.5% sevoflurane till the 6th and 13th day for 2h, respectively. 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 20 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 14days 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 sevoflurane on morphology of DCs: Observing with inverted microscope, before exposured to sevoflurane, the precursor cells are small and the suface is glossy .The size of three group cells are obviously lagger than the precursor cells till 3or4 days with clusters emerging. The surface became irregular and has petty dendritic pseudopods around till 5or6 days.After exposured to sevoflurane,when observed the 14th day,the dendritic pseudopods arounding cells of Group I are shorter and fewer,compared with Group C.Contrarily, the dendritic pseudopods arrounding cells of GroupM are longer and denser, similar to Group C. The surface observed with scanning electron microscope(SEM)is coincident with the results above.2 Effects of sevoflurane on the expression of CD80/CD86 and CD83/ HLA–DR:Compared with Group C,the expresstion of CD80/CD86 and CD83/ HLA–DR of GroupI has decreased (P<0.05) and the result was 19.64±2.57%, 7.12±1.45%,but there is no statistical significance between Group M and Group C(P>0.05).3 Effects of DCs on the proliferation of the allogeneic mixed lymphotyte: The mature DCs have a stronger capable to stimulate the mixed lymphocyte to proliferate but the immature could not or weaker. Group I exhibited weaker activity in stimulating the proliferation of allogeneic mixed lymphocyte because the cells were in the immature stage as compared with group C and M (P<0.05). Group C and group M could stimulate the mixed lymphocyte to proliferate very strongly but there is no statistical significance between Group M and Group C(P>0.05).4 Effects of sevoflurane on the level of the IL–12 production: Compared with Group C, the IL– 12 production of Group I decreased(P<0.05)and the result was 377.14±311.51pg/ml. There is no significant difference in IL-12 levels between GroupC and GroupM (P>0.05).Conclusions: Sevoflurane can inhibit the maturation of imDCs from cord blood mononuclear ,but it has show little influence to vatilis of mDCs.