摘要目的 观察经骨髓间充质干细胞条件培养基培养的退变髓核细胞的生物学活性及细胞外基质表达的变化.方法 (1)从成年SD大鼠股骨和胫骨中提取骨髓间充质干细胞,传代培养后,收集骨髓间充质干细胞条件培养基;(2)取成年大鼠,破坏其椎间盘纤维环,2周后获得椎间盘退变大鼠模型,提取大鼠脊柱髓核细胞;(3)以骨髓间充质干细胞条件培养基培养髓核细胞24 h.结果 与正常髓核细胞比较,退变髓核细胞的活性[(59.43±12.33)％,t=11.647,P＜0.01]明显降低,细胞中Ⅱ型胶原(0.50 ±0.12,t=14.677,P＜0.01、0.49 ±0.17,t =7.153,P＜0.01)、蛋白聚糖(0.72 ±0.23,t=15.436,P＜0.01、0.26±0.32,t=8.816,P＜0.01)蛋白及mRNA表达水平明显减少,基质金属蛋白酶-3(1.48 ±0.31,t=15.436,P＜0.01、2.00±0.24,t=29.640,P＜0.01)蛋白及mRNA表达水平明显增多.而骨髓间充质干细胞条件培养基可显著恢复退变髓核细胞的活性(79.62±10.11,t=4.786,P＜0.01),并促进细胞中Ⅱ型胶原(1.23 ±0.16,t=10.933,P＜0.01;0.74±0.16,f=3.506,P＜0.01)、蛋白聚糖蛋白(1.49 ±0.28,t=9.359,P＜0.01;0.99±0.37,t=6.846,P＜0.01)及mRNA表达,降低基质金属蛋白酶-3(0.49 ±0.39,t=9.359,P＜0.01;0.89±0.12,t=21.934,P＜0.01)蛋白及mRNA表达,但骨髓间充质干细胞条件培养基对正常髓核细胞的活性(94.87±8.36,t=0.524,P＞0.05)以及细胞中Ⅱ型胶原(1.52±0.18,t=0.299,P＞0.05;1.21 ±0.31,f=1.543,P＞0.05)、蛋白聚糖(1.81 ±0.32,t=0.486,P＞0.05;0.99 ±0.16,t=0.094,P＞0.05)和基质金属蛋白酶-3(0.24 ±0.14,t=0.486,P＞0.05;0.55±0.11,t=0.593,P＞0.05)蛋白及mRNA表达水平没有明显影响.结论 骨髓间充质干细胞条件培养基对退变髓核细胞的增殖以及细胞外基质蛋白表达有促进作用.

abstractsObjective To observe the biological activities and expression of extracellular matrix of degenerative nucleus pulposus cells which were cultured in the bone marrow mesenchymal stem cells conditioned medium.Methods Bone marrow mesenchymal stem cells were extracted from the femur and tibia of adult SD rats.After subculture,the conditioned medium of bone marrow mesenchymal stem cells was collected.We destroy the annulus fibrosus of the adult rats,and the intervertebral disc was obtained 2 weeks later.Degenerative rat model was used to extract rat nucleus pulposus cells.The nucleus pulposus cells were cultured in the bone marrow mesenchymal stem cells conditioned medium for 24 hours.Results Compared with normal nucleus pulposus cells,the activity of degenerative nucleus pulposus cells [(59.43 ± 12.33) ％,t =11.647,P ＜ 0.01] was significantly decreased,and type Ⅱ collagen in cells (0.50 ± 0.12,t=14.677,P＜0.01;0.49 ±0.17,t=7.153,P＜0.01),proteoglycan (0.72 ±0.23,t=15.436,P ＜0.01;0.26 ±0.32,t =8.816,P ＜0.01) protein and mRNA expression levels were significantly reduced,the expressions of matrix metalloproteinase 3 protein and mRNA were significantly increased (1.48 ± 0.31,t =15.436,P ＜ 0.01;2.00 ± 0.24,t =29.640,P ＜ 0.01.The conditioned medium of bone marrow mesenchymal stem cells can significantly restore the activity of degenerative nucleus pulposus cells (79.62 ± 10.11,t =4.786,P ＜ 0.01),and promote type Ⅱ collagen in cells (1.23 ± 0.16,t =10.933,P ＜0.01;0.74 ±0.16,t =3.506,P ＜0.01),proteoglycan protein (1.49 ±0.28,t =9.359,P ＜0.01;0.99 ±0.37,t =6.846,P ＜0.01) and mRNA expression,matrix metalloproteinase 3 protein and mRNA expression was decreased (0.49 ± 0.39,t =9.359,P ＜ 0.01;0.89 ± 0.12,t =21.934,P ＜0.01),but the activity of conditioned medium of bone marrow mesenchymal stem cells on normal nucleus pulposus cells (94.87 ± 8.36,t =0.524,P ＞ 0.05) and type Ⅱ collagen in cells (1.52±0.18,t =0.299,P＞0.05,1.21 ±0.31,t=1.543,P＞0.05),proteoglycan (1.81 ±0.32,t =0.486,P ＞ 0.05,0.99 ± 0.16,t =0.094,P ＞ 0.05) and matrix metalloproteinase 3 (0.24 ± 0.14,t =0.486,P ＞ 0.05,0.55 ± 0.11,t =0.593,P ＞ 0.05) Protein and mRNA expression levels were not significantly affected.Conclusion The bone marrow mesenchymal stem cell conditioned medium promoted the proliferation of extramedullary nucleus cells and the expression of extracellular matrix proteins.