骨髓间充质干细胞对退变髓核细胞功能的逆转作用

目的 研究骨髓间充质干细胞(BMSCs)对退变髓核细胞(dNPCs)的营养修复作用。方法 针刺抽吸法制备SD大鼠退变椎间盘动物模型,分离培养髓核细胞(NPCs)及BMSCs,并将BMSCs和dNPCs分别复合藻酸盐后等比例接种于transwell共培养系统上下室中进行体外非直接接触共培养作为实验组,单纯NPCs和dNPCs分别复合藻酸盐后作为阳性及阴性对照组。光镜下观察细胞生长情况,选取合适细胞复合藻酸盐在共培养体系中培养7 d后回收各组NPCs,对回收细胞的CollagenⅠ、Ⅱ和Aggrecan行Western blot及免疫荧光检测,RT-PCR检测CollagenⅡ、SOX9和Aggrecan的mRNA的表达情况。结果 共培养7 d后,RT-PCR检测结果显示BMSCs+dNPCs组的Collagen Ⅱ、SOX9和Aggrecan的mRNA表达接近NPCs组,但明显高于dNPCs组(P均＜0.05)。Western blot检测结果显示BMSCs+dNPCs组的Collagen Ⅱ和Aggrecan含量接近甚至超过NPCs组,但明显高于dNPCs组(P均＜0.05);CollagenⅠ含量与NPCs组相近,但明显低于dNPCs组(P＜0.05)。Collagen Ⅱ和Aggrecan免疫荧光染色结果显示,BMSCs+dNPCs组和dNPCs组胞质都被染成红色,在核周围着色较深,BMSCs+dNPCs组胞质着色范围及核周围深染范围明显大于dNPCs组。结论 在Transwell非直接接触共培养条件下,BMSCs能够促进dNPCs外基质的表达,提示BMSCs能够在一定程度上逆转椎间盘dNPCs的功能。

Role of Bone Marrow Mesenchymal Stem Cells in Restoring the Functions of Degenerative Nucleus Pulposus Cells

Objective To explore the effects of bone mesenchymal stem cells(BMSCs) in restoring the functions of degenerative nucleus pulposus cells(dNPCs).Methods The animal models of degenerative nucleus pulposus were established by means of acupuncture and aspiration.The BMSCs as well as the normal and degeneratived nucleus pulposus cells of SD rats were isolated and cultured.The BMSCs/alginate gel complex and dNPCs/alginate gel complex were used for indirect co-culture in vitro,which was set as experiment group.The NPCs and dNPCs cultured alone as positive and negative controls.The cell growth conditions were observed by light microscopy,and suitable cells were selected to combine alginate gel stents and cultured in transwell plate.Seven days later,nucleus pulposus cells of each group were recycled,and the mRNA expressions of CollagenⅡ,SOX 9,and Aggrecan were detected by RT-PCR,and the CollagenⅠ,Ⅱ,and Aggrecan were detected by Western blotting and immunofluorescence.Results After non-contact co-culture for 7 days,the mRNA levels of CollagenⅡ,SOX 9,and Aggrecan increased apparently in BMSCs+dNPCs group,while it was significantly lower in dNPCs sample(all P＜0.05).The content of CollagenⅡand Aggrecan detected by Western blotting in BMSCs+dNPCs group got close to NPCs sample,but it was significantly higher than dNPCs samples(all P＜0.05),while the content of CollagenⅠin BMSCs+dNPCs group got close to NPCs samples,but it was significantly lower than dNPCs sample(P＜0.05).Immunofluorescence results showed that cytoplasm was dyed red and the color near the caryon became dark in BMSCs+dNPCs group and dNPCs group;however,the colored scope of cytoplasm and the dark colored scope surrounding the caryon in BMSCs+dNPCs group was obviously larger than dNPCs group.Conclusion Under a 3D non-contact co-culture system,BMSCs can promote the expression of epimatrix of the dNPCs,which shows that BMSCs can restore the functions of dNPCs of intervertebral disc to certain extent.