CDMP-1逆转人退变椎间盘髓核细胞的实验研究

Effects of CDMP-1 on human nucleus polus cells in the disc degeneration

[目的]通过CDMP-1促进退变髓核细胞外基质合成来逆转椎间盘退变。[方法]分离、培养人退变椎间盘髓核细胞,取第3代髓核细胞,应用CDMP-1分组进行干预实验。A组为对照组,B组和C组为实验组,B组加入100 ng/ml CDMP-1,C组加入200 ng/ml CDMP-1。采用光学相差显微镜及透射电镜对对照组和实验组细胞形态和超微结构进行对比观察:XTT-PMS法检测各组髓核细胞12 d的生长曲线,研究三组细胞间的生长动力学差异;实时荧光定量PCR检测髓核细胞,7、14、21 dⅡ型胶原和糖胺多糖的mRNA表达。[结果]光学相差显微镜及透射电镜示:两剂量实验组形态学上没有明显差异,与对照组相比,CDMP-1实验组能较好的维持髓核细胞生物形态学特性,延长退变髓核细胞的衰老;三实验组12 d的生长曲线一致,两剂量的CDMP-1没有增加退变髓核细胞的增殖;实时荧光定量PCR示100 ng/ml CDMP-1组:Ⅱ型胶原mRNA表达总体均数及3个时间点均高于另外两组任一时间点(P<0.05);糖胺多糖mRNA表达总体均数及14、21 d 2个时间点均高于另外两组相应时间点(P<0.05),且Ⅱ型胶原和糖胺多糖mRNA表达量随刺激时间增加而增加。200 ng/ml CDMP-1组:提高了糖胺多糖mRNA表达水平的同时,却降低了Ⅱ型胶原mRNA水平。[结论]100 ng/ml的CDMP-1可以延长体外培养人退变髓核细胞的衰老,维持细胞生物形态学特性,提高退变髓核细胞Ⅱ型胶原和糖胺多糖mRNA表达水平,在一定程度逆转了椎间盘髓核细胞退变。

[Objective]To reverse the disc degeneration through improving the extracellular matrix synthesis of the nucleus polus cells by CDMP-1. [Methods]Human degenerated intervertebral disc cells were harvested and then cultured in the mediums,degenerated intervertebral disc cells of passage 3 were randomly divided into three groups.Group A：the control group,group B：with 100 ng/ml CDMP-1 incubated,group C：with 200 ng/ml CDMP-1 incubated.All cells were observed with light microscopy and transmission electron microscopy for gross morphology and ultrastructure.To investigate the growth kinetic difference between groups,the growth curres of the cells were detected for 12 days by XTT-PMS method.And the expressions of type Ⅱ collagen and GAG in degenerated intervertebral disc cells were measured by real time RT-PCR at 7,14 and 21 days,separately.[Results]In light microscopy and transmission electron microscopy observations,it showed nogross morphology difference between the CDMP-1 groups.But in comparison with the control group,the CDMP-1 groups imdicated postponed aging of degenerated intervertebral disc nucleus pulposus cells.And all groups exhibited the same growth curves line during the 12 days.This indicated that CDMP-1 in different concentrations did not simulate the proliferation of the degenerated intervertebral disc cells.In the 100 ng/ml CDMP-1 in different concentrations the mRNA syntheses in GAG and type Ⅱ collagen were significantly increased on real time RT-PCR.But in the 200 ng/ml CDMP-1 group,the GAG mRNA expression was increased,while the mRNA expression of collagen type Ⅱ was reduced.[Conclusion]CDMP-1,in concentrations of 100 ng/ml,can delay the aging of degenerated intervertebral disc nucleus pulpous cells in vitro,maintain the characteristics of the gross morphology,improve the mRNA expressions of GAG and type Ⅱ collagen,and reverse the degeneration of intervertebral disc cells to some extent.