正常与退变髓核细胞对骨髓间充质干细胞诱导分化的对比研究

Comparative study of degenerated versus non-degenerated nucleus pulposus cells in inducing mesenchymal stem cells differentiation

目的正常髓核细胞(nNPCs)对骨髓间充质干细胞(MSCs)有诱导分化作用,而退变髓核细胞(dNPCs)对MSCs的诱导作用是否存在差异尚未阐明,文中对比nNPCs与dNPCs对MSCs向髓核细胞分化诱导作用的差异。方法细胞株/退变患者来源的人NPCs与细胞株来源人MSCs行体外三维共培养,分为5组:nNPCs对照组、dNPCs对照组、MSCs对照组、nNPCs-MSCs组(nNPCs诱导的MSCs)、dNPCs-MSCs组(dNPCs诱导的MSCs),共培养7 d后,行RT-PCR及Western blot检测各组细胞聚集蛋白聚糖(ACAN)、Ⅱ型胶原(COL-2)、SOX-9、基质金属蛋白酶-1(MMP-1)、MMP-3的表达水平。结果 RTPCR结果显示,与nNPCs对照组相比:dNPCs对照组ACAN、COL-2、SOX-9的表达较低(P<0.05),MMP-1、MMP-3的表达相对较高(P<0.01);nNPCs-MSCs组ACAN的表达相对较低(P<0.05);dNPCs-MSCs组ACAN、COL-2、SOX-9的表达相对较低(P<0.05)。与dNPCs对照组相比:nNPCs-MSCs组ACAN、COL-2、SOX-9的表达相对较高(P<0.05),MMP-1、MMP-3的表达较低(P<0.01);dNPCs-MSCs组MMP-1、MMP-3的表达较低(P<0.01)。与MSCs对照组相比:nNPCs-MSCs组及dNPCs-MSCs组ACAN、COL-2、SOX-9的表达均相对较高(P<0.01)。与nNPCs-MSCs组相比:dNPCs-MSCs组ACAN、COL-2、SOX-9的表达相对较低(P<0.05)。Western blot结果显示:与nNPCs对照组相比:dNPCs对照组ACAN、COL-2、SOX-9的表达较低(P<0.05),而MMP-1、MMP-3的表达较高(P<0.05);nNPCs-MSCs组MMP-1、MMP-3的表达相对较低(P<0.01);dNPCs-MSCs组ACAN、COL-2、SOX-9、MMP-1、MMP-3的表达较低(P<0.05)。与dNPCs对照组相比:nNPCs-MSCs组ACAN、COL-2、SOX-9的表达较高(P<0.05),MMP-1、MMP-3的表达较低(P<0.01);dNPCs-MSCs组MMP-1、MMP-3的表达较低(P<0.01)。与MSCs对照组相比:nNPCs-MSCs组及dNPCs-MSCs组ACAN、COL-2、SOX-9的表达均相对较高(P<0.01)。与nNPCs-MSCs组相比:dNPCs-MSCs组ACAN、COL-2、SOX-9的表达均相对较低(P<0.05)。结论 nNPCs及dNPCs均能诱导MSCs向髓核样细胞分化,与nNPCs共培养后,MSCs的ACAN、COL-2、SOX-9的表达水平与nNPCs相当,优于同样培养条件下dNPCs对MSCs的诱导。

Objective Normal nucleus pulposus cells （nNPCs） can induce the differentiation of mesenchymal stem cells （MSCs）. However, the inductive effect of degeneration nucleus pulposus cells （dNPCs） on MSCs has not been determined. This study aimed to compare nNPCs with dNPCs in inducing MSCs into nucleus pulposuslike cells.Methods MSCs （cell line） were co-cultured with NPCs （cell line/isolated from patients with intervertebral disc degeneration） in 3D in vitro. The cells were divided into 5 groups： nNPCs control group, dNPCs control group, MSCs control group, nNPCs-MSCs group （MSCs induced by nNPCs）, dNPCs-MSCs group （MSCs induced by dNPCs）. After co-cultured for 7 days, the expression level of aggrecan （ACAN）, type II collagen （COL-2）, SOX-9, matrix metalloproteinase-1 （MMP-1） and MMP-3 in all the 5 groups’ cellswere detected by RT-PCR and Western-blot.Results The RT-PCR Results showed the expression of ACAN, COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs-MSCs group （all P〈0.05）. The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in dNPCs control group （all P〈0.01）, but there was no difference when compared with nNPCs control group and MSCs control group （all P〉0.05）. Western-blot analysis showed that the expression of ACAN, COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs-MSCs group （all P〈0.05）. The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in nNPCs control group and dNPCs control group （all P〈0.01）, but there was no difference compared with MSCs control group （all P〉0.05）.Conclusion Both nNPCs and dNPCs could induce the differentiation of MSCs into nucleus pulposus cells. After co-cultured with nNPCs, the expression level of ACAN, COL-2 and SOX-9 of MSCs was similar to that of nNPCs, which was superior to the induction of MSCs by dNPCs under the same culture conditions.