| Objective (l)To dicuss the suitable condition of inducing inmature dendritic cells(DC) in vitro.(2)To discuss the feasibility of immune tolerance in rat small bowel transplantation inducedt by immature dendritic cells isolated from SD and Wistar rat.(3)To discuss the difference of immunological tolerance in rat small bowel transplantation induced by immature dendritic cells come from donor and recipient. (4)To discuss the difference of immunological tolerance in rat small bowel transplantation induced by immature dendritic cells come from liver and bone marrow.Methods: Dendritic cells isolated from liver or bone marrow of inbred strain Wistar and SD rat were cultured with 10ng/ml granulocyte macrophage colony stimulating factor (GM-CSF) and/or transforming growth factor beta one (TGFβ1) for five to nine days. DCs were observed with light microscope and scan electron microscope during culturing time.The expressing of their immune phenotypes (CD80,CD86, MHC II)were analyzed by FCM on the fifth, seventh and ninth days. The ability to atimulate T cells of DCs were assayed by 3-(4,5-dimethylthiazol-z-y)-2, 5-diphemylte trozalium bromide (MTT) .A week begore operation, the recipient rats were injected 2×10~6 inmature DCs from peripheral vein and observed. Then establish the models of heterotopic small bowel transplantation (SBT: SD→ Wistar).According to the different pretreatment in recipients, animals were divided into 6 groups. Group A : SD→Wistar SBT; Group B: SD→Wistar SBT + inject immature DC isolated from SD rat liver and culture with rGM-CSF. Group C: SD—Wistar SBT + inject immature DC isolated from SD rat bone marrow and culture with rGM-CSF. Group D: SD—Wistar SBT + inject immature DC isolated from SD rat bone marrow and culture with rGM-CSF ,TGF pi . Group E: SD—Wistar SBT + inject immature DC isolated from Wistar rat liver culture with rGM-CSF. Group B: SD—-Wistar SBT + inject immature DC isolated from Wistar rat bone marrow culture with GM-CSF(r) ,TGF pi.In vitro generation of DCs, dendritic cells (DC) were isolated from rat liver or from bone marrow. These DCs were incubation of GM-CSF and/or TGF pi. Then the expression levels of MHC II and CD80, CD86 on DC were detected, and the ability of inducing immune response of DC was also analvzed.After SBT, the recipient were observed such things as body weight reaction , abdominal sign, survival time and so on. A part of graft's small bowll ,about 2 centimetres long, were resected and sterilized by formalin fume on the first postoperation day (POD1), third postoperation day (POD3) , fifth postoperation day (POD5), seventh postoperation day (POD7). Acute immuno-rejection levels were evaluated by HE.Among six groups,the results of rejection levels were compared by analysis of Radit. Survival rate was calculated by product-limit estimates. Survival curve was calculated and compared by log-rank test. Kaplan-Meier curve was drew and the median survival time was calculated based on the curve.P<0.05 is the significance level.Results: Under light microscope, DCs were were observed suspending in complete RPMI 1640 medium during the first two days. The cells wre small and round without dendritic process. About after seven days, nonadherent cells were observed. On the 9th day , the cultured cells become round and irregular and exhibit the typical morphological features of DC .The suspending cells were examined with scanning electronic microscope . On the 5th day ,DCs displayed typical morphology with elongated dendritic processes .A number of immature DC were congregating or scattering here and there. The cells had different size .The cell membrane looks like the brim of skirt .In the early phase ,every inmature DC has the appearance of bloomy peony .These mature DCs like nerve cell and extend many processes.After cultured and inducted for five or seven days , DCs were identified by FCM and MTT. Inmature DCs expressed surface antigens in low levels .including CD80 (<10%) ,MHC II (<15%), CD86 (<2%) The immunological function assayed by MTT showed that DC did not have the ability to stimulate vigorous proliferation of allo-T cells . After culture and induction for nine days , mature DCs expressed high level surface antigens .including CD80( >70% ),CD86( >50%),MHC II (>70%). The immunological function assayed by MTT showed that DCs had the ability to stimulate vigorous proliferation of allo-T cells.Heterotopic small bowel transplantation in rat was a success in 51 cases and fail in 9 cases with the successful rate being 85.0%. 33 recipients survived over seven days. The longest survival time was 14 days. The average survival time was 8.5 days. On POD3, POD5, POD7,the graft rejection in A group is the most serious and B group is the most mild.The total operation time was 130±30 minutes. The operation time of donors was 45±15 minutes. The operation time of recipient was 60±40 minutes. The anastomosis time of arteria was 20±15 minutes. The anastomosis time of vein was 3O±35 minutes.Recipients in each group were able to move, drink in normal when the anesthesia was over. On POD1 and POD2, the mucosa of stoma was red and moisten in every group. Mesentery artery in recipient graft pulsated well .The wall of bowel was intact and had no tension . Killed the rat and check the bowel, it is found that the graft was good withoutadhesion , ischemia , necrosis and node swell. Rat in A group on POD3, E and F group on POD4 , B and D group on POD7 express few activity , bad appetite , reducing of body weight, thick mucus of stoma. Stoma in A group on POD6 was more narrow than before. The bowel appeared purplish red and necrosed. Hair of rats looks untidy. On the ninth day, graft became pale , bowel dilated and go together in a mass. Matter like cheese filled in bowel tube. Mesentery artery in recipient graft pulsated weak. Such phenomena appeared later in B and D group.The median survival time is 12.5 days in D group, 10.5 days in B group, 7.0 days in F group, 6.7 days in E group, 6.0 days in C group, 5.3 days in A group. Graft rejection was observed from past operation3 day(POD3) on by histopathology. On POD3 ,the Graft rejection in A group is more severe than that in B,D group. On POD5, the Graft rejection in A group is more severe than that in B, D, E, F group, and D group is more severe than that in C group. On POD7, the Graft rejection in A, C group is more severe than that in B,D group.Conclusion:l.GM-CSF and TGFpM are strong immune depressive cytokine which can affect the proliferation and differentiation of DC, prevent the maturity of DC.2.1nmature DC can induce allogenic immunological tolerance of rat small bowel transplantation.3.The degree of tolerance induced by inmature DC isolated from liver and bone marrow is different. The immature DC isolated from liver can prolong the survival time of rat small bowel graft after transplantation and alleviate the severity of graft rejection more than that of inmature DC isolated from bone marrow. If put TGFpi into the medium , the DC isolated from bone marrow can prolong the survival time of rat small bowel graft after transplantation and alleviate the severity of graft rejection again.4. The degree of tolerance induced by inmature DC isolated from donorand recipient is different. The immature DC isolated from donor canprolong the survival time of rat small bowel graft after transplantationand alleviate the severity of graft rejection more than that of inmature DCisolated from recipient .5.This research provides an important approach to control immunerejection in small bowel transplantatin and may be enhancing apply ofsmall bowel transplantatin in patients.
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