| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 4255857 | Transplantation Proceedings | 2015 | 6 Pages |
•We isolated and identified Kupffer cells of donor rats.•We successfully constructed a B7 RNA interference vector, which we used to reduce B7 expression in the donor Kupffer cells.•Suppression of B7 expression in donor Kupffer cells can significantly reduce the activation of recipient T lymphocytes and secretion of IL-2, and may induce immune tolerance of liver transplants.
BackgroundKupffer cell (KC), a kind of important antigen-presenting cell in liver, play an important role in the process of acute rejection after liver transplantation. The aim of this study was to investigate effect of suppression of donor KC B7 expression on recipient lymphocyte activation and secretion of interleukin-2 (IL-2) in vitro.MethodsLiver ex vivo perfusion with collagenase IV and density-gradient centrifugation were used to isolate donor Lewis rat KCs. The interference fragments of the B7 molecule were designed to construct RNA interference vector pSilencer 3.1H1-Neo-B7 that was transfected into KCs of donor rat. Reverse-transcription polymerase chain reaction was used to detect the changes in the expression of B7 molecules in KCs. The transfected KCs were divided into 3 groups: A, control group; B, empty vector group; and C, RNA interference group. The lymphocytes of recipient Brown Norway (BN) rats were isolated and cocultured with the cells in the 3 groups. Enzyme-linked immunosorbent assay was used to detect the content of IL-2 in the culture supernate. Methylthiazolyl tetrazolium assay was used to detect the proliferation of lymphocytes.ResultsThe yield rate of KCs was 5 × 107, and the cell viability was >98%. RNA interference vector had been successfully constructed and identified by means of enzyme digestion and sequencing. The expression of B7 in KCs decreased by 22% after RNA interference (P < .01). After coculturing with lymphocytes of BN rats, compared with the control group, the decreased expression of B7 significantly inhibited the activation and proliferation of lymphocytes as well as the secretion of IL-2 by lymphocytes. The proliferation of lymphocytes in recipient BN rats decreased by 49% (P < .01), and the secretion of IL-2 in the culture supernate decreased by 67% (P < .01).ConclusionsThis study successfully constructed a B7 RNA interference vector, and applied it to assessing reduction of B7 expression in donor KCs. RNA interference significantly suppressed the activation of recipient T lymphocytes and secretion of IL-2 via the CD28/B7 costimulatory pathway and may induce immune tolerance in liver transplants.
