强直性脊柱炎患者骨髓间充质干细胞的生物学及免疫学特性

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

目的：探讨强直性脊柱炎（AS）患者骨髓间充质干细胞（BMSCs）的生物学及免疫学特性，为进一步阐明AS的发病机制和寻找新的治疗靶点提供理论依据。方法：选取37例活动期AS患者（AS组），男34例，女3例，平均年龄（24.3±5.4）岁，HLA-B27均为阳性；49例健康志愿者作为对照组（HD组），男43例，女6例，平均年龄（25.7±4.9）岁；其中HLA-B27阴性44例（HD1组），HLA-B27阳性5例（HD2组）。从每例受检者髂后上棘穿刺采集骨髓组织，分离BMSCs，培养扩增至第3代，以1×104/ml的浓度接种于96孔板中，100μl/孔，从第1天开始每日取3孔进行细胞计数，共计12d，绘制生长曲线；每日取3孔经MTT处理后测定吸光度值，绘制细胞活力曲线，观察BMSCs的生物学特性。使用流式细胞仪检测各受检者BMSCs的细胞表面表型。将第3代BMSCs细胞接种于U形底96孔板，培养4h后使用60Co照射30Gy；取健康志愿者外周血采用密度梯度法分离单个核细胞（PBMCs），加入细胞培养液，计数后按BMSCs∶PBMCs 1∶20、1∶10、1∶5、1∶2、1∶1的比例接种于已接种BMSCs的96孔板，共培养5d，观察双向混合淋巴细胞反应情况；同样获取PBMCs，计数后同样以5个比例接种于96孔板，加入植物血凝素（PHA）4μg/ml，充分接触后共培养5d，观察淋巴细胞增殖反应情况。对组间进行统计学比较。结果：AS组、HD1和HD2组第3代BMSCs体外培养1~12d时的增殖能力、细胞活力无显著性差异（P〉0.05）；三组细胞表面表型均为高水平表达CD105、CD73和CD90，不表达CD45、CD34、CD14和HLA-DR。HD1组和HD2组BMSCs与不同比例PBMCs共培养的双向混合淋巴细胞反应和PHA刺激的淋巴细胞增殖反应均无显著性差异（P〉0.05）；AS组与HD组比较有显著性差异（P〈0.05）。结论：AS患者BMSCs的生物学特性无明显改变，但其免疫调节功能明显下降，其可能在AS的发病机制中扮演重要角色。

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.