Proteomic analysis in serum of rat hind-limb allograft tolerance induced by immunosuppressive therapy with adipose-derived stem cells

Background: Adipose-derived stem cells combined with transient immuno-suppression prolonged vascularized composite tissue allotransplant survival and induced immune tolerance in a rodent hind-limb model. The authors investigated serum proteins in the adipose-derived stem cell tolerance group and control group using proteomic study. Methods: An orthotopic hind-limb model from Brown-Norway to Lewis rats was used. The control group received no treatment. Rats in the tolerance group received combined treatments of short-term cyclosporine A, antilymphocyte serum, and multiple rounds of adipose-derived stem cells. Serum samples were analyzed. Spots of interest were subjected to in-gel trypsin digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to elucidate the peptide mass fingerprints. The mass spectrometric characteristics of the identified proteins were analyzed. Immunohistochemical analysis of transplanted tissue and enzyme-linked immunosorbent assay of serum were validated. Results: Rats in the tolerance group had significantly higher amounts of β2-glycoprotein, α1-macroglobulin, rat-albumin, and vitamin D-binding protein, and significantly lower levels of haptoglobin compared with controls. Immunohistochemical staining of the alloskin indicated similar effects, such as upregulated vitamin D-binding protein and down-regulated haptoglobin in the tolerance group compared with rejection controls (p < 0.05). Enzyme-linked immunosorbent assay revealed that vitamin D-binding protein was statistically increased (p < 0.05) and haptoglobin expression was significantly decreased (p < 0.01) in the tolerance group compared with the controls. Conclusions: There were significant differences in the serum proteomics between the tolerance and control groups. Down-regulated haptoglobin and up-regulated vitamin D-binding protein are involved in adipose-derived stem cell-induced immune tolerance and allotransplant survival.