| OBJECTIVE To investigate the feasibility and possible mechanism of immune tolerance induced by low-dose of FK506 combined with intraportal transplantation of mesenchymal stem cells (MSC).MATERIALS AND METHODS The model of orthotopic liver transplantation (OLT) was established and evaluated in rats based on the modified Kamada's two-cuff technique. The key point of rat liver transplantation is to shorten non-hepatic period. Some skillful modified measures should be used in this orthotopic liver transplantation model.MSCs of male SD rat were cultured in medium, and labeled with 5, 6- carboxyfluorescein diacetate, succinimidyl ester (CFDA). Then, the marked MSC were injected into portal vein of the rats. The liver graft were sliced up and dyed with 4, 6-diamidino-2- phenylindole (DAPI) seven days after transplantation, and observed under fluorescent microscope.Thirty two OLTs models were established by modified Kamada two-cuff technique, with inbred female Brown Norway BN)rats and female LEW rats served as recipient and donor respectively. The recipients were randomized into 4 groups. Group A: rejection group received intraportal injection normal saline (LEWIS-BN,n = 8); Group B: MSC transplantation group (LEWIS-BN,n = 8) underwent intraportal transplantation of MSC (2.0×106 per rat); Group C: FK506 treatment group (LEWIS-BN,n = 8),lavaged with FK506 (0.5mg/kg.d-1) from post-transplant day1 to day13; Group D: Combined treatment group underwent intraportal transplantation of MSC (2.0×106 per rat) and accepted subtherapeutic immunosuppression (FK506 0.125 mg/kg.d-1) for 13 days(LEWIS-BN,n = 8). Of six recipients in each group were feeded, and observed the animal survival days. Levels of serum ALT and TB were assayed, and the histopathologic changes in livers were evalued with routine methoeds after OLT 2 weeks. Flow cytometry was used to measure the expression of TITC-anti-HIS19 in BN splenocytes so as to observe the SRY gene detection in peripheral blood.RESULTS Compared with the control group ((12.5±0.8) d) and MSC transplant alone group ((12.7±1.2) d), the survival period was significantly longer in animals of MSC transplant combined with low dose FK506 treated group ((52.8±4.1) d, P < 0.01), almost same long as that of in whole dose FK506 treated group ((47.8±3.8) d). The scores of liver allografts decreased markedly in recipients with subtherapeutic FK506 treatment combined MSC intraportal transplantation (Gradeâ… ) as compared to that in animals in control group (gradeâ…¢) and MSC transplant alone group (gradeâ…¡) , based on Banff evaluation system. The rate of anti-HIS19 positive splenocytes in animals of MSC transplant combined with low dose FK506 treated group ((13.0±2.5) %) was significant higher than that in control group ((0.4±0.5) %) and MSCs transplant alone group ((0.5±0.3) %), as well as in whole dose FK506 treated group ((1.4±0.9) %, P < 0.01). Moreover, the serum concentrations of AST were significantly decreased in animals of MSC transplant combined with low dose FK506 treated group ((49.48±9.70) U /L) and whole dose FK506 treated group ((54.3±11) U /L), as compared with that of in control group ((886.81±90.3) U /L, P < 0.01) and MSC transplant alone group ((869.7±109) U /L, P < 0.01) on post- transplant day 14. As compared to control group and MSC transplant alone group, the levels of serum TBIL in animals of MSC transplant combined with low dose FK506 treated group and whole dose FK506 treated group were also remarkedly decreased(P < 0.01) at the end time point.CONCLUSIONS: MSCs could protect the function of graft liver from severe rejection. Combined administration of low-dose FK506 and allogeneic MSC might prolong recipient survival days, due to chimerism formation and immune tolerance inducing.
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