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

为了研究间充质干细胞对NP细胞体外和体内活化的影响，我们将NP细胞与间充质干细胞（MSCs）共培养。方法：兔椎间盘提取NP细胞。采用12孔复培养板共培养。采用膜培养插入物。将NP细胞以1.0 X 10^4个细胞/孔的速度接种到每孔中，膜培养插入物中有或没有1.0 X 10^4个MSCs。共培养第7天，采用WST-8法评估细胞增殖情况，通过测定[3H]-胸苷的摄取来评估DNA合成，通过测定[35S]-硫酸盐的摄取来评估蛋白多糖（PG）的合成。统计学分析采用Wilcoxon's sign -rank检验，显著性设为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。椎间盘退变在复位后2、4、8和16周进行评估。结果：在细胞增殖试验中，插入MSCs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）仅为4.02 X 10^4个细胞/孔（p<0.01）。M组[3H]-胸腺嘧啶的平均崩解速率（DPM）为188.56±5.20 DPM /细胞，N组为9.54±0.65 DPM /细胞（p<0.01）。[35S]-硫酸盐在M组为64.16±1.66 DPM /细胞，在N组为6.42±0.15 DPM /细胞（p<0.01）。这些结果与体内研究的结果相吻合。结论：髓核细胞与间充质干细胞共培养比单层培养更能促进细胞增殖和基质合成；髓核细胞经间充质干细胞激活后，用共培养的方法重新插入髓核细胞，在减缓椎间盘退变方面比我们以往的研究更有效。

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.