Studying On The Immunotolerance Induced By Extracorporeal Photopheresis Of The Human Body

In this study,psoralen combined with A-band ultraviolet(PUVA),also known as extracorporeal photopheresis(ECP),induced apoptosis of spleen lymphocytes(SP),and then Immature dendritic cells(imDC)co-cultured with apoptotic splenic lymphocytes to induce the formation of novel dendritic cells,called extracorporeal photochemotherapic dendritic cells(ecpDC)).To investigate the effect of ecpDC on lipopolysaccharides(LPS)-mediated dendritic cells(DC)maturation,and to investigate whether ecpDC is suitable for use in human body Immune tolerance function,ecpDC for liver transplantation in patients with induced immune tolerance to provide theoretical support and experimental basis.First,human peripheral blood mononuclear cells(PBMC)were isolated from human peripheral blood mononuclear cells(PBMC).Recombinant human granulocyte colony stimulating factor(recombinant human interleukin 4,rhIL-4)Human granulocyte-macrophage colony stimulating factor,rhGM-CSF).(PUVA-SP)obtained by PUVA method,and the apoptosis rate was detected.The immunohistochemical dendritic cells(ecpDC)were co-cultured with imDC and PUVA-hSP.PUVA-SP,SP and im DC were co-cultured,harvest ecp DC and SPDC.Collecting imDC,ecpDC and adding 100 ng / mL LPS for 1 d,respectively,to obtain LPS-stimulated imDC and LPS-stimulated ecp DC,ie imDC + LPS group,ecpDC + LPS group.The expression of CD11 c,CD83 and CD86 on the surface of the cells was detected.The supernatant was obtained and the levels of IL-10 and IL-12 cytokines were measured by ELISA.The proliferation of syngeneic T cells was detected by mixed lymphocyte culture of imDC,DC and ecpDC.The results showed that:(1)After treatment with PUVA,the total apoptosis rate was(98.59 ± 1.35)% and(3.64 ± 0.73)% in the control group,while the early apoptotic rate of PUVA treatment group was(94.21 ± 3.75)% Higher than the control group(P <0.01).The positive expression rate of CD83 and CD86 in SPDC group was(45.86 ± 4.86)%,(42.47 ± 0.76)%,which was significantly higher than that in imDC group(15.06 ± 0.59)% and(15.19 ± 1.83)%(P <0.01).The positive expression rate of CD83 and CD86 in the ecpDC group was(22.83 ± 5.26)%,(22.06 ± 4.37)% and the expression rate of CD83 and CD86(15.06 ± 0.59)% and(15.19 ± 1.83)% in imDC group(99.79 ± 0.36)% and(99.85 ± 0.19)%,respectively(P <0.01).The expression of CD83 and CD86 in DC group was significantly higher than that in DC group(P <0.01).The concentration of IL-12 in supernatant of imDC group was(147.52 ± 6.42)pg / ml,which was higher than that of ecp DC group(134.92 ± 13.54)pg / ml(P <0.05)(326.86 ± 7.58)pg / ml(P <0.01).The concentration of IL-10 in the supernatant of imDC group was(51.66 ± 5.72)pg / ml,which was significantly higher than that in ecpDC group(172.61 ± 12.12)pg / ml(P <0.01)The concentration of IL-10 in the supernatant was(121.19 ± 10.62)pg / ml,which was significantly lower than that in the ecpDC group(172.61 ± 12.12)pg / ml(P <0.01).The expression rates of CD83 and CD86 were(99.79 ± 0.36)% and(99.85 ± 0.19)% in imDC + LPS group,which was significantly higher than that in ecpDC + LPS group(35.15 ± 5.40)%,(32.66 ± 2.66%)(P <0.01).The concentration of IL-10 in the supernatant of imDC group was(51.66 ± 5.72)pg / ml,which was lower than that of ecpDC group(172.61 ± 12.12)pg / ml(P <0.01),imDC + The concentration of IL-10 in LPS group was(121.19 ± 10.62)pg / ml,which was lower than that of ecpDC + LPS group(316.09 ± 30.91)pg / ml(P <0.01).5,imDC group stimulation index of 1.817 ± 0.094,DC group stimulation index of 3.460 ± 0.071,ecpDC group stimulation index of 1.422 ± 0.060.Compared with imDC group,DC cells could significantly stimulate T cell proliferation,and there was significant difference between the two groups(P <0.01).Compared with imDC group,ecpDC group could significantly inhibit the proliferation of T cells,both of which had significant statistics Differences(P <0.01).Through the above results we can draw the following conclusions: First of all,this study uses in vitro photochemical therapy to induce early apoptosis of splenic lymphocytes,for the field of research provides a new method,compared with other methods are easy to operate,high security features.Followed by imDC cells phagocytosis of apoptotic splenic lymphocytes obtained by the method of new DC cells,that is,ecpDC,expression of immature phenotype and can inhibit the recipient T lymphocyte proliferation reaction,confirmed that ecpDC has a similar negative immune regulation with Dreg The Finally,ecpDC differs from imDC in that ecpDC is resistant to LPS-mediated stimulation of dendritic cell maturation.It can be used to induce immune tolerance in vivo and not easily activated as mature DC cells.More suitable for use in the human body to induce immune tolerance.