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

For the current medical, organ transplantation is one of the most important ways for saving the loss of organ function or failure of the patient’s life of Organ failure. As the only effective way for the treatment of end-stage liver diseases,liver transplantation has been developed rapidly over recent years. At present, many types of immunosuppressive drugs have used to prevent and suppress immune rejection after liver transplantation,and the immunosuppressive regimens are various;but great variety of immunosuppressive drugs, have significant side-effects presently. Extracorporeal photopheresis(ECP)has been approved he treatment of cutaneous T-cell lymphoma and has demonstrated its benefits for graft-vs-host disease(GVHD), and recurrent solid-organ allograft rejection. ECP is a new effective way to control postoperative rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treatedex vivowith8-methoxypsoralen and UVA light radiation(PUVA).The mechanisms of ECP therapy are unclear, but accumulating evidence suggests that the immunoregulatory properties of ECP therapy include modulation of APCs and induction of regulatory T cells(Tregs).In vivo production of immunosuppressive cytokines by monocytes and macrophages after ingestion of autologous apoptotic cells has been suggested as an important immunomodulatory effect of ECP.Previous studies focused only on ECP treatment of recipient immune cells.Our study is the first to extend the target of ECP treatment to donor immune cells.UVA with8-MOP can significantly induce the apoptosis of human spleen lymphocytes,especially the early apoptosis, by inducing the dendriticcells by rh GM-CSF、IL-4 with the peripheral blood of the transplantaion recipients.they can be used as a novel negative immunoregulatory tool,which would provide further support to transplant immune tolerance in future.1.UVA combined with 8-MOP induced the apoptosis of human spleen lymphocytes. First,gain the spleen from the donor liver transplantation(the spleen derived from recipient of liver transplantation of Department of Hepatobiliary Surgery, Institute of organ transplantation, 309 th Hospital of PLA.The source of donor liver have signed the informed consent and approved by the ethics committee of309 Hospital,comply with the provisions of relevant transplantation series of medical ethics).First, the human spleen were harvested from liver allograft donors to get the suspension of lymphocytes, which were divided into four groups( UVA group,8-MOP group,UVA+8-MOP group and control group)according to different treatments. 3 cases of each group, UVA group at cultivated in 37℃5%CO2 incubator. And putting in 50 ml / L, the irradiation intensity for 2J / cm2 light radiometer in irradiation and irradiation for 9 min, the UVA light source distance is20 cm; 8-MOP group were adding the 200 ng / ml 8-MOP and then cultivated in 37℃5%CO2 incubator incubation for 20 min; UVA+8-MOP group were adding the 200 ng / ml 8-MOP first,then cultivated in 37℃5%CO2 incubator incubation for 20 min,after that they were putting in 50 ml / L, the irradiation intensity for 2J / cm2 light radiometer in irradiation and irradiation for 9 min;the blank control group, only to pure the culture of human spleen lymphocytes.Then the different treatment of human spleen lymphocyte were suspened with RPMI1640 medium 1200r/min centrifugation for 5 min, repeated washing for two times, then RPMI 1640 complete culture medium(containing 100 ml / L fetal bovine serum) cells resuspended in suspension and is placed at37℃5%CO2 incubator incubation,After 24 hours, by annexin V-FITC/PI apoptosis detection reagent box in each group were stained and by flow cytometry after different treatment of human spleen lymphocyte apoptosis rate. Finally,detecting the human spleen lymphocytes apoptosis rate by the flow cytometry.2.The peripheral blood of liver transplantation donor preparation of immature dendritic cells:First,gain the peripheral blood of liver transplantation donor about 10ml(the blood derived from recipient of liver transplantation of Department of Hepatobiliary Surgery, Institute of organ transplantation, 309 th Hospital of PLA.The source of donor liver have signed the informed consent and approved by the ethics committee of 309 Hospital, comply with the provisions of relevant transplantation series of medical ethics).Putting in the two 15 ml centrifuge tube,marked induction group and control group.Add the equal amount of human lymphocyte separation medium of peripheral blood,isolating the mononuclear cells,Then add 10 ml RPMI1640 after washing two times,under the microscope to count the number of living cells, the viability of cells>95%.The cell concentration was adjusted to1 × 106 /L,then were inoculated in 6 well culture plate,the treated lymphocytes were cultivated in 37℃5%CO2 incubator for 2h,then removal the suspended cells,the two groups were added in rh GM-CSF(500u/ml) 2ul, rh IL-4(1000u/ml)and 8ul,then were changing half of the medium after 48 h, adding rh GM-CSF(500u/ml) 1ul, rh IL-4(1000u/ml)and 4ul, induced group remained unchanged after 96 h, the control group added rh TNF- alpha 3ul, cultured for 2 days. In 6 days of incubation, collecting their adherent cells. marked CD80,CD83,CD11 a and CD86 antibodies by PE or FITC, phenotypic analysis of DC on flow cytometry.Results :1. UVA combined with 8-MOP induced the apoptosis of human spleen lymphocytes:The early apoptosis rate of human spleen lymphocytes processed by the UVA+ 8-MOP group was(98.45±0.54) %,significantly higher compared with that of UVA group(P<0.01)and 8-MOP group(P<0.01). 8-MOP group was( 64.68±0.74) %,which is significantly higher than the UVA group(61.05±0.90)%(P<0.01)and control group(13.67±0.54)%(P<0.01).Both UVA group and 8-MOP group induced the early apoptosis rate were higher than the control group(P<0.01). The total apoptosis rate of human spleen lymphocytes processed by the UVA+ 8-MOP group was(99.36±0.39)%, The early apoptosis rate is(98.45±0.54)%,significantly higher compared with the other three groups. The total apoptosis rate and the early apoptosis rate of each group were detected:the total apoptosis rate and apoptosis rate of the combined group were(99.36±0.39)%,(98.45 ± 0.54)%; the total apoptosis rate and apoptosis rate of 8-MOP group were(69.55 ±0.46)%,(64.68 ±0.74)%; the total apoptosis rate and early apoptotic rate of UVA group were(65.42 ±0.36)%,(61.05 ±0.90)%; the total apoptosis rate and early apoptosis rate of the control group were(15.53 ± 0.45)%,(13.67 ±0.54)%.2.The peripheral blood of liver transplantation donor preparation of immature dendritic cells: In the 6d, induced group to detect the content of CD80 is(22.63 ±3.56) %, CD86 content is(16.39 ±4.72)%,CD83 is(8.58 ±1.72)%, CD11 c is(70.56 ±10.22)%,accord with the performance of im DC. In the 6d, the control group culture to detect the content of CD80 is(53.82 ± 5.46)%, CD86 is(46.65 ±6.35)%, CD83 is(80.56 ±16.45)%, CD11 c is(82.32 ±11.61)%, accord with the performance of m DC. Therefore, on the 6d,the induction group deteced the content of CD80, CD86, CD83, CD11 c were significantly lower than that of the control group(P < 0.01), and under invertedmicroscope to show the status of the im DC.And the two groups of indexes accord with the state, with the rh GM-CSF+rh IL-4 culture method can be successfully used in peripheral blood of human cultured im DC.By using the ECP as the treatment,a large quantity apoptotic cells have been induced. Moreover, the lymphocytes of early apoptokotic stage have shown significantly higher than treatment group and the control group. A great number of im DC have been incubated with combination of rh GM-CSF and rh IL-4. and cells presented mature cellular structure of DC through inverted microscope. they can be used as a novel negative immunoregulatory tool,which would provide further support to transplant immune tolerance in future.