| The immunosuppression therapy has been improved greatly nowadays, but acute rejection is still a major cause for renal allograft loss. Early diagnosis and treatment are crucial to the restoration of allograft function after renal transplantation. Though fine-needle biopsy is the golden standard for accurate diagnosis, noninvasive methods are still preferred by many clinicians and patients. With the development of molecular biology, the role of cytokines in rejection is now paid much more attention. Now it's hot to study the cytokine and cytotoxic molecule gene expressions and to intervene them during acute rejection.It is well known that IL-2/IL-2R plays an important role during acute renal allograft rejection. In both human and animal experiments, anti-IL-2R antibody remarkably reduces the acute rejection rate, but there is still a portion of subjects who developed acute rejection. The facts implied there are other pathways which independent of IL-2/IL-2R system participated in acute rejection. IL-15 is produced by nonlymphoid cells including monocytes, dendritic cells and bone marrow stromal cells; it is a growth factor and an activator of CD8 memory T cells and was observed up-regulation during early liver acute rejection. Perforin and granzyme B are produced by CTL during acute rejection to attack the allograft. Although there are some reports about the perforin and granzyme B up-regulation during acute rejection of organ transplantation, the relationship between IL-15 and these two cytotoxic molecules are still unknown. In this study, we investigated the role of IL-15 in renal allograft rejection and the expressions of IL-15, perforin and granzyme B mRNA during renal acute rejection in both human and rats. The thesis is divided into three parts: Part I: Establishment of a modified model of renal transplantation in the rat0bjective : To establish a simple and reliable model of heterotopic renal allograft transplantation in rats.Providing a more practical and high acute rejection rate model for experimental studies of renal transplantation.Methods:Using healthy adult WISTAR rats as donor and SD rats as recipient, Donor's vena cava and a patch of aorta attached to renal artery was anastomosis end-side to the recipient's vena cava and abdominal aorta. Ureter with bladder's vane was suture to the recipient's bladder.Results:The average total time of operation was (90±10) min and the successful rate was 86%.Conclusions:This is a feasible and reliable renal transplantation model with high acute rejection rate which is suitable for the study of organ transplantation mechanism in rats.Part II: The study of the efficacy of blockading IL-15 to reduce the cytotoxic lymphocyte activity of acute renal allograft rejection in the ratObjective To study the role of IL-15 in the process of acute rejection(AR) and investigate the efficacy of blockading IL-15 on the recipients to reduce the expressions of cytotoxic attack molecules granzyme B (GraB) and perforin (PFP) during acute renal allograft rejection in rats.Methods Male Wistar rats and SD rats were used as donors and recipients, heterotopic renal transplantation model was established and four groups (n≥15/group) were involved in this study: (1). isograft group was used as the control group (Ctrl); (2). acute rejection group (AR) (received no treatment); (3). CsA treatment group (CsA) (received CsA 6mg.kg-1.d-1, i.p. posttransplant); (4) antibody treatment group (AB) (received anti IL-15 antibody 0.5mg/kg, i.p. on day 0, 2, 4 and 6 posttransplant). Animals were sacrificed on day 1, 3, 5 and 7 after transplantation. Allograft specimens and blood were collected. Allograft tissues were analyzed by pathologic assay and the expressions of IL-15, PFP and GraB mRNA in serum as well as renal tissues were detected by real-time polymerase chain reaction (real-time PCR).Results (1). The expressions of IL-15, PFP and GraB mRNA were increased in groups AR, AB and CsA in both serum and tissue, respectively; compared to the Crtl group. The expressions reached highest points on day 3 or 5 and decreased on day 7, respectively; (2). In AR group, the expressions of IL-15, PFP and GraB mRNA were significantly higher in both serum and tissue than that of the other groups (P<0.01), and increased distinctly 3 days after transplantation, which about 2-3 days earlier than the appearance of clinical symptoms. (3). The expression of IL-15 mRNA in AB group was significantly lower compared to AR group and CsA group (P<0.05 ) . (4). The pathological results showed that the severe AR occurred on the 7th day after transplantation in group AR, respectively; whereas the rejection level were much mild in AB group. Ctrl group and CsA group showed no sign of AR.Conclusions These data demonstrate that (1). The expressions of IL-15, PFP and GraB up-regulated early in the serum as well as tissue in rat renal AR model; IL-15 participated in the early stage of AR and the pathway maybe different from IL-2; (2). The up-regulation of PFP and GraB is related to the up-regulation of IL-15 in the early stage of AR; (3). Blocking IL-15 in the early stage of AR can down-regulate the expressions of PFP and GraB thus would have a promising results in control the progression of cytotoxic lymphocyte activity of acute renal allograft rejection in the rat; (4). Using real-time PCR to evaluate expressions of PFP,GraB and IL-15 may be a non-invasive as well as sensitive method to make an early diagnosis of acute allograft rejection and monitor the efficacy of anti-rejection therapy. Our findings might refine existing anti-rejection strategies. Part III: Expressions of IL-15, perforin and granzyme B in peripheral blood during acute rejection of kidney transplantationObjective To evaluate the value of IL-15, PFP and GraB in the early diagnosis and differential diagnosis of acute rejection(AR) after kidney transplantation. Methods Forty-five recipients of renal allograft were included in the study. The expressions of PFP, GraB and IL-15 mRNA were detected by SYBR Green I real time quantitative polymerase chain reaction (FQ-PCR).Results The expressions of IL-15, PFP and GraB mRNA were increased significantly more in patients with AR (n=9) than those with stable renal function (n=23) and were about 2 to 3 days earlier than the appearances of clinical AR symptoms. After steroid therapy, all the AR was reversed and the expressions of PFP, GraB and IL-15 mRNA were decreasing at the same time. The expressions of PFP and GraB mRNA were also increased in cases of bacteria infection(n=8)but IL-15 mRNA remained unchanged. After the treatment of the infections, the expressions of PFP and GraB mRNA were down-regulated. The expressions of PFP, GraB and IL-15 mRNA had no significant difference in CsA-induced nephrotoxicity (n=5) during the study.Conclusions Sequential monitoring the expressions of PFP, GraB mRNA combined with IL-15 mRNA in serum of renal allograft recipients can be as useful markers for the early diagnosis of AR and differential diagnosis from bacterial infection and it is valuable to observe the therapeutic effectiveness of AR after kidney transplantation. FQ-PCR had high sensitivity and reproducibility, it is suitable for clinical practice.
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