Preliminary Study On Tolerance Induction Of Rat Metanephros Anlagen By Combined Xenotransplantation Of Thymus Anlagen

It had been demonstrated that E14 and E15 metanephroi allografted into adultSD rats without using immunosuppressives resulted in revascularization andorganogenesis of kidney primordia, and exhibited excretory function. However,rejection remains a principal obstacle to organogenesis of metanephroi allograft andxenograft. Although E16 and E17 metanephroi alltransplanted into Cyclosporine-treated hosts showed better outcome, the non-specific immunosuppressive agentscontinuously used would lead host to susceptibility to neoplasms and infection.Immune tolerance study is now the hot topic in transplantation community.Because it can continuously provide the negative thymus selection, the central-typedimmune tolerance was though to be a most powerful T cell tolerance type, andwould be a most promising approach.Allo- or xenotransplantation tolerance can be induced by thymustransplantation, which is another way to induce the central type immune tolerancebased on the specific host antigens. In this study, it was investigated whether theimmunologic function can be reestablished and isogenesis heterogenic donorimmune tolerance can be induced by combined transplantation of embryonic thymusanlagen in athymia nude mice. Materials and MethodsMale and female rats were mated overnight, and female rats were scored forvaginal plaques in the next morning. The presence of vaginal plaques was taken torepresent embryonic day 0(E0). Whole fetal metanephroi and thymus anlagen fromembryonic day 16(E16) Lewis rats were taken to perform the present experiment.Lewis E16 thymus and metanephros primordia were get out from embryo andimmediately preserved at 4℃in normal saline and then were implanted into theabdomen of adult athymus mice.Host nude mice were randomly divided into four groups: GroupⅠ: E16 Lewismetanephroi+thymuses→Nude mice;Ⅱ: E16 Lewis metanephroi→Nude mice;GroupⅢ: E16 Lewis thymuses→Nude mice;Ⅳ: None of E16 Lewis metanephroi+thymuses→Nude mice. The paired E16 metanephroi taken from Lewis rat wereimplanted into the omentum of Nude mice and the primordia of thymus into kidneycapscules.3 and 5 weeks post-implantation, the peritoneotomy was carried out on therecipients, metanephros and thymus transplants were removed for histopathologicalexamination.The skin graft was performed 6 weeks post transplantation. The skin of Lewisrats,BN rats,C57BL and Nude mice were transplanted in the chest-abdomen of theⅠ,Ⅱ,ⅢandⅣgroups mice. 7 days post-implantation, the change of the skinswere examined once a day. Look on the skin flap became black more than 80% orformed a scab as rejection. Count the time of skin flap survived.12 weeks post-implantation, the spleen cells were obtained from nude mice formixed lymphocyte reacion(MLR) test,and the blood of nude mice was obtained todetect re-establishment of T lymphocyte with flow cytometry(FCM) . Mixed lymphocyte reacion(MLR) was performed by taking spleen cell suspension ofLewis,BN and C57BL mice to prepare stimulation cells; and the single cellsuspension extracted from recipients spleens ofⅠ,Ⅱ,ⅢandⅣgroups act asresponse cells. Then, OD values were measured with enzyme-labeled analysator,The record was expressed as stimulate index(SI); SI=the OD values of allograftgroup/ the OD values of substratum group.Results3 and 5 weeks post-implantation, well-developed E16 thymus transplant wereobserved in each group with numerous incomplete lobules subdivided byinterlobular septa, and lobules divided into two parts, cortices, which werecomposed of densely packed lymphocytes, and medullas, which were composed ofloosely packed lymphocytes. E16 Lewis metanephros grafts had well structure.Metanephroi had enlarged, and were soft and kidney-like, become vascularized.Both cortical and medullary tissues were present. Cortices contained well-developedglomeruli containing red blood cells, proximal tubules with well-developed brushborder membranes, and distal tubules. Rare accumulations of lymphocytes wereobserved. Medullas contained well-developed collecting ducts..5 weeks post-implantation, Survivle time of receptor was observated afternephrectom.. groupⅠrecipients survived longer than groupⅣ(p<0.01) andgroupⅡ(p<0.05), groupⅡlonger than groupⅣ(p<0.05).6 weeks post-implantation, skin survival time of Lewis, BN rats and C57BL/6(H-2~b) mice transplanted into nude mice of different groups were observed. Skinsurvival time of BN rats and C57BL/6 (H-2~b) mice in groupⅠandⅢare shorterthan groupⅡandⅣ. Skin survival time of Lewis in groupⅠandⅢis longer thanthat of BN rats and C57BL/6 (H-2~b) mice. 12 weeks post-implantation, MLR indicate that the groupⅠandⅢhad normalreaction to spleen cells of the BN rats and C57BL/6 (H-2~b) mice. SI in groupⅠorⅢnude mice against Lewis rat was obviously lower . FCM indicate that thepercentage of CD3 ~+CD4~+ T cells inⅠ,Ⅲgroup receptors step up obviously,almost approach to the percentage of CD3 ~+CD4~+T cells in C57BL/6 (H-2~b) miceblood.Conclusions1. E16 Lewis thymus and kidney anlagen allografted into nude mice did grow,differentiate and undergo organogenesis persistently and function well.2. Nude mice with E16 thymus anlagen of Lewis rats implanted could reestablishimmunologic function and specificly response to antigen from donor origin.3. E16 Lewis thymus and kidney anlagen xenografted into nude mice well growand develop in the abdomin of the nude mice and may inducexenotransplantation tolerance with our protocol.