兔ADSCs与NPCs共培养后再移植对椎间盘退变影响

THE EFFECTS OF REIMPLANTATION OF CO-CULTURED ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AND NUCLEUS PULPOSUS CELLS ON INTERVERTEBRAL DISC DEGENERATION IN RABBIT

目的探讨兔脂肪间充质干细胞(ADSCs)与髓核细胞(NPCs)共同培养后再植入兔腰椎间盘对Ⅱ型胶原和蛋白多糖的影响。方法体外分别提取分离培养兔ADSCs与NPCs。诱导分化兔ADSCs向成骨、成脂方向分化,鉴定兔ADSCs。将兔ADSCs与NPCs共同培养,诱导兔ADSCs向NPCs方向分化。新西兰大白兔30只,分为实验组和对照组,分别将兔ADSCs与NPCs共同培养后的细胞悬液、PBS缓冲液注入针刺抽吸后的椎间盘。4、8周后,采用间苯三酚分光光度法检测两组兔椎间盘髓核蛋白多糖表达的变化,采用反转录-聚合酶链反应对Ⅱ型胶原和蛋白多糖的基因表达进行半定量分析。结果 ADSCs可诱导向成骨、成脂方向分化,与NPCs共同培养后由梭形、多角形变为成纤维细胞样。对照组髓核Ⅱ型胶原和蛋白多糖表达低于实验组,差异有显著性(t=6.50～13.04,P<0.05)。结论兔脂肪组织可成功提取分离ADSCs,ADSCs与NPCs共同培养后移植可延缓兔椎间盘退变。

Objective To study the reimplantation of co-cultured adipose derived mesenchymal stem cells （ADSCs） and nucleus pulposus cells （NPCs） into intervertebral disc （IVD）, and observe its effect on type collagen and proteoglycan in rabbits. Methods Rabbit ADSCs and NPCs were isolated and cultured in vitro. The ADSCs were induced and differentiated to the direction of adipogenic and osteogenic lineages. ADSCs and NPCs were co-cultured and induced to differentiate into NPCs. Thirty New Zeal- and white rabbits were devided into experimental group and control group, co-cultured cell suspension of the ADSCs and NPCs, as well as PBS buffer solution, was injected into the discs after aspirated by a needle. Four and eight weeks later, the changes of proteoglyean in NPCs were detected by spectrophotometrie method, semiquantitative analysis was done using reverse transcription po- lymerase chain reaction for gene expressions of type Ⅱ collagen and proteoglyean gene. Results ADSCs could be induced to dif- ferentiate into adipogenic and Osteogenic lineages. Spinedle or multi-angular ADSCs turned into fibro-like phenotype in the co-cul- ture with NPCs. The expressions of type Ⅱ collagen and proteoglycan in the control group were weaker than that in the experimen- tal group （t=6.50-13.04,P〈0.05）. Conclusion ADSCs can be successfully extracted and isolated from rabbit adipose tis- sue, transplantation of co-cultured stem cells and NPCs can delay the degeneration of intervertebral discs in rabbits.