Basic research on decelerating disc degeneration -Activation of nucleus pulposus cells-

将骨髓间充质干细胞（mesenchymal stem cells，MSCs）与髓核细胞（NP）共培养，观察MSCs对髓核细胞体外和体内活化的影响。方法：从兔椎间盘中收获NP细胞。使用12孔多重培养板进行共培养。使用膜培养插入物。将NP细胞以1.0 X 10^4个细胞/孔接种到每个孔中，在膜培养插入物内有或没有1.0 X 10^4个MSC。在共培养的第7天，通过使用WST-8评价细胞增殖，通过测量[3 H]-胸苷的摄取评价DNA合成，通过测量[35 S]-硫酸盐的摄取评价蛋白聚糖（PG）合成。使用通过Wilcoxon符号秩检验的统计分析，显著性设定为p<0.01。然后将共培养的NP细胞重新插入兔椎间盘退变模型中。在重新插入后2、4、8和16周评价椎间盘退变。结果如下：在细胞增殖试验中，插入物内接种MSC的NP细胞（M组）为2.97 X 10^5个细胞，而单层培养的NP细胞（N组）仅计数4.02 X 10^4个细胞/孔（p<0.01）。[3 H]-胸苷的平均每秒崩解（duration per second，duration），M组为188.56±5.20 μ g/细胞，N组为9.54±0.65 μ g/细胞（p<0.01）。[35 S]-硫酸盐的结果显示M组为64.16±1.66 μ M/细胞，N组为6.42±0.15 μ M/细胞（p<0.01）。这些结果与体内研究的结果良好相关。结论：与单层培养相比，间充质干细胞与髓核细胞的共培养导致细胞增殖和基质合成的改善，并且使用共培养方法激活的髓核细胞的再植入比我们先前的研究更有效地减缓椎间盘退变。

NP cells were cocultured with mesenchymal stem cells (MSCs) in order to evaluate the effect of mesenchymal stem cells on activation of NP cells in vitro and in vivo. Methods : NP cells were harvested from rabbit discs. A 12-well multiple culture plates were used for coculture. A membrane culture insert was used. NP cells were seeded into each well at 1.0 X 10^4 cells / well, with or without 1.0 X 10^4 MSCs inside a membrane culture insert. On day 7 of coculture, they were evaluated for cell proliferation by using WST-8, DNA synthesis by measuring the uptake of [3H]-thymidine and proteoglycan (PG) synthesis by measuring the uptake of [35S]-sulphate. Statistical analysis by Wilcoxon's signed-ranks test was used with significance set at p<0.01. Cocultured NP cells were then reinserted into disc degeneration models in rabbits. Disc degeneration was evaluated at 2, 4, 8 and 16 weeks post reinsertion. Results : In cell proliferation assay, NP cells seeded with MSCs inside the insert (group M) was 2.97 X 10^5 cells, where as NP cells cultured monolayer (group N) only counted 4.02 X 10^4 cells / well (p<0.01). The average disintegration per second (DPM) measured for [3H]-thymidine indicating DNA synthesis was 188.56±5.20 DPM / cell in group M and 9.54±0.65 DPM / cell in group N (p<0.01). Results for [35S]-sulphate demonstrated 64.16±1.66 DPM / cell in group M and 6.42±0.15 DPM / cell in group N (p<0.01). These results correlated well with the results of in vivo studies. Conclusions : Coculture of nucleus pulposus cells by mesenchymal stem cells resulted in an improvement of cell proliferation and matrix synthesis over monolayer culture and reinsertion of nucleus pulposus cells activated by mesenchymal stem cells using coculture method was more efficient than our previous studies in deceleration of intervertebral disc degeneration.