非接触共培养条件下骨髓间充质干细胞向类髓核细胞的诱导分化

Effect of indirect co-culture system on differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells

背景:组织工程中间充质干细胞治疗椎间盘退变性疾病为退变椎间盘结构和功能恢复提出了一种新型的治疗方案,是目前研究的热点。目的:分析兔骨髓间充质干细胞向类髓核细胞分化的潜能。方法:将第3代兔骨髓间充质干细胞与兔髓核细胞按培养要求分为3组:普通6孔板单纯培养骨髓间充质干细胞组,普通6孔板单纯培养髓核细胞组,骨髓间充质干细胞与髓核细胞共培养组,Transwell 6孔板底部接种第3代骨髓间充质干细胞,上层接种第3代髓核细胞。倒置显微镜观察各组细胞形态的变化,在培养第7天采用免疫细胞化学、RT-PCR及Western blot技术测定各组细胞Ⅱ型胶原和聚集蛋白聚糖的表达。结果与结论:(1)共同培养第7天,骨髓间充质干细胞呈多角形、短梭形改变,无纤维样变化,形态接近髓核细胞;(2)共培养组骨髓间充质干细胞Ⅱ型胶原和聚集蛋白聚糖的表达与单独培养髓核细胞组相近,显著高于单独培养骨髓间充质干细胞组;(3)结果表明,非接触共培养条件下,髓核细胞具有诱导骨髓间充质干细胞向类髓核细胞分化的潜能,为椎间盘退变性疾病的生物学治疗提供大量的细胞来源。

BACKGROUND：Mesenchymal stem cel s （MSCs） for the treatment of degenerative disc diseases present a new therapeutic strategy for the structural and functional recovery of the degenerative intervertebral disc. OBJECTIVE：To study the differentiation potential of rabbit bone marrow MSCs （BMSCs） into nucleus pulposus-like cel s. METHODS：Passage 3 BMSCs and nucleus pulosus cells （NPCs） extracted from rabbits were assigned into simple BMSCs culture, simple NPCs culture or co-culture group. In the former two groups, BMSCs or NPCs were cultured in 6-well culture plates. In the co-culture group, passage 3 BMSCs and NPCs were cultured on the upper or lower layer of the 6-well Transwell chamber, respectively. Cell morphology was observed by inverted contrast phase microscope. At 7 days of culture, immunocytochemistry, RT-PCR and western blot were used to examine the expression levels of type II collagen and aggrecan. RESULTS AND CONCLUSION：At 7 days of co-culture, BMSCs were polygonal or short spindle-shaped, with no fiber-like changes, and cel morphology was close to that of NPCs. Additional y, the expression levels of type II col agen and aggrecan in the BMSCs co-cultured with NPCs were similar to those in the NPCs cultured alone, but significantly higher than those in the BMSCs culture only. Overal , these results show that coculture of BMSCs with NPCs can induce BMSCs differentiating into an NPCs phenotype, indicating that BMSCs may provide sufficient sources of cel s for the biological treatment of degenerative disc diseases.