Biological characteristics and immunologic properties of bone marrow-derived mesenchymal stem cells in ankylosing spondylitis

English Keywords:Ankylosing spondylitis  Bone marrow-derived mesenchymal stem cells  Biological characteristics  Immunologic properties

English Abstract:

  【Abstract】 Objectives: To study the biological characteristics and immunomodulation properties of bone marrow-derived mesenchymal stem cells(BMSCs) in ankylosing spondylitis(AS), and to offer theoretical basis for clarifying the pathogenesis and searching new therapy target of AS. Methods: 37 AS patients(3 females and 34 males) with an average age of 24.3±5.4 years in active stage and 49 healthy donors(HDs) (6 females and 43 males) with an average age of 25.7±4.9 years were included in the study. All of the AS patients were HLA-B27-positive; conversely, 44 healthy donors were HLA-B27-negative(HD1) and 5 healthy donors were HLA-B27-positive(HD2). The bone marrow samples were obtained from every AS patient and healthy donor by posterior superior iliac spine aspiration. BMSCs were separated, cultured in vitro to the third generation (P3), and seeded in 96-well plates at a concentration of 1×104/ml, 100μl/well. Three wells of each sample were digested for cell counting per day up to 12 days. The BMSCs growth curves were made using the data for cell proliferation obtained above. Using MTT that was to determine absorbance at 490nm, the cell viability curves for BMSCs were also acquired. The surface markers of BMSCs were assayed using flow cytometry. Briefly, BMSCs were seeded in U-bottomed, 96-well culture plates for 4 hours for adherence, and then irradiated(30Gy) with 60Co before being cultured with the mixed PBMCs in 2-way MLR or the PBMCs stimulated by PHA in the lymphocytes proliferation reaction. PBMCs were obtained by the Ficoll-Hypaque gradient separation of the buffy coat of HDs. The mixed PBMCs were then mixed with different amounts(1∶20, 1∶10, 1∶5, 1∶2, 1∶1, BMSCs∶PBMCs) of BMSCs in the 96-well plates co-cultured for 5 days, 2-way MLR was studied. Compared with the MLR, the allogeneic PBMC proliferation assay only used one allogeneic PBMC from a healthy volunteer stimulated with PHA, co-cultured with BMSCs for 5 days, lymphocytes proliferation reaction was also studied. The results of groups were analysed with statistics. Results: There was no any significant difference about proliferation ability and cell viability of P3 BMSCs cultured ex vivo 1-12 days among the AS patients, healthy donors 1 and healthy donors 2(P>0.05). They all expressed high levels of the surface markers CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 and HLA-DR surface molecules. But, in either the MLR or the PBMC proliferation assay stimulated with PHA, the 3H-TdR assay and MTT assay data suggested a statistically significant reduced immunomodulation potential of BMSCs from AS patients, compared with the healthy donors(P<0.05), the difference between HD1 and HD2 was not significant statistically(P>0.05). Conclusions: Biological characteristics of BMSCs from patients with AS do not change, but the immunomodulation properties of BMSCs reduced in AS, and the BMSCs with reduced immunomodulaiton potential may be involved and play a novelty role in pathogenesis of AS.