In vitro skin transplantation and TUNEL assay in evaluation of cellular immunity caused by infusion of rhesus mesenchymal stem cells / 第二军医大学学报

ABSTRACT

Objective: To transfuse rhesus bone marrow derived mesenchymal stem cells (MSCs) to rhesus leucocytes antigen (RhLA) mismatched recipients, in attempt to study the immunological reaction between recipients' T lymphocytes and the skin of donor or a third party. Methods: Bone marrow aspirates were collected from femurs of donor rhesus, and the mononuclear cells were isolated and resuspended in low glucose Dulbecco's Modified Eagles Medium (DMEM) containing 2% fetal bovine serum. Phenotype of MSCs was analyzed by flow cytometry and the differentiated cells were identified by relevant specific staining. Allogeneic MSCs were given to recipients (single infusion) without preconditioning. Peripheral blood lymphocytes from recipients were co-cultured in vitro with the donor's skin or a third rhesus for 72 h before and 20 days, 40 days, and 60 days after infusion. The pathological lesions were evaluated and the keratinocyte apoptosis of the skin sections was determined by TUNEL assay. Results: Twenty days after infusion, the pathological lesion in in vitro skin explants from MSCs donor was less and the apoptosis rate was declined to (12.2±1.9)% from a pre-infusion level of (23.0±4.9)%. The apoptosis of cells in skin explants from the third party also declined to (11.8±1.4)% from a pre-infusion level of (22.0±2.0)% (P<0.05). Intensive immunological reaction was observed between recipient and donor/the third party on day 40 and day 60 after transplantation, with the apoptosis rate being (22.0±3.2)%, (22.0±2.5)% (donor) and (23.2±2.4)%, (24.0±2.6)% (the third party). Conclusion: Allogenic MSCs transfusion can decrease the immunological reaction between donors and recipients in rhesus, but this decreased reaction is temporary and reversible.