脂肪间充质干细胞向髓核样细胞诱导分化的初步研究

Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro

[目的]定向诱导大鼠脂肪间充质干细胞分化为髓核样细胞,为椎间盘退变性疾病的细胞移植治疗奠定生物学基础。[方法](1)200gSD雄性大鼠断颈处死后,取腹股沟脂肪垫处脂肪,0.1%胶原酶消化,体外接种培养,同时采用极限稀释法纯化细胞;(2)利用流式细胞技术对细胞表面标记进行鉴定;(3)第3代细胞消化计数后,用1.2%海藻酸钠溶液悬浮,调整细胞密度至1×106/ml,滴入3.5%CaCl2溶液形成微球,加入含TGF-beta1的诱导培养基,低氧条件下进行微球联合培养;(4)7d后,取部分微球进行AlcianBlue染色并收集微球内细胞提取RNA,进行RT-PCR检测。[结果](1)体外培养的脂肪间充质干细胞主要呈梭形和多角形,核大、核仁明显,平行排列生长或呈漩涡状生长;(2)流式细胞仪检测显示Sca-1、CD44的阳性率分别为95.2%、99.7%,CD45、CD11b的阳性率分别为0.4%、1.5%;(3)微球联合培养7d后,Alcian Blue染色表明,实验组细胞外基质的生成明显多于对照组;(4)RT-PCR结果证实,髓核细胞标志基因和软骨细胞标志基因的表达,实验组明显高于对照组。[结论](1)本实验方法可以获得大量均一的脂肪间充质干细胞;(2)脂肪间充质干细胞可以被定向诱导分化为髓核样细胞;(3)首次探讨了将脂肪间充质干细胞体外诱导后移植治疗椎间盘退变性疾病,为椎间盘退变性疾病的干细胞移植治疗提供了新的思路。

[ Objective] To differentiate rat adipose tissue-derived mesenehymal stem cells （ADSCs） into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based translantation therapy of degenerated interverte： bral discs. [ Method] Rat ADSCs were isolated from the subcutaneous inguinal region, and purified using the method of limited dilution. The third passages were analyzed by fluorescence activated cell sorter （ FASC ） to examine the cell surface markers （Sca-l, CD44, CD45 and CD11 b）. To induce ADSCs towards a nucleus pulposus-like phenotype, the author immobilized ADSCs in 3-dimentional alginate hydrogels and cultured them in a inducing medium containing transforming growth factor-betal （TGF-betal） under hypoxia （2% 02 ）, while control groups were under nomorxia （21% 02 ） in alginate beads in medium with or without TGF-betal. Semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） was carried out to evaluate expression of characteristic genes in the process＇of differentiation. Meanwhile, Alcian blue staining was used to detect the formation of sulfated glycosaminoglycans （GAG） by the differentiating cells. [ Result ] The purified ADSCs by limited-dilution appeared fibroblast-like shapes and proliferated rapidly in vitro. Results of flow cytometry showed that ADSCs were positive for Sca- 1 and CD44 and negative for CD45 and CD11 b. The outcome of RT-PCR manifested that the gene expression of Sox-9, aggrecan and collagen type II, which were chondrocyte specific, were upregulated in medium containing transforming growth factorbetal under hypoxia （2% O2 ） ; likewise, gene expression of HIF-1a, which was characteristic of intervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the large formation of GAG. [ Conclusion] （ 1 ） The approach in this experiment proved to be a simple and effective way to acquire a large quantity of homogenous ADSCs. （2） Rat ADSCs can be differentiated into nucleus pulposus-like ceils. （ 3 ） For the first time, this paper discussed the way of regenerating degenerated intervertebral discs using ADSCs by firstly differentiating them in vitro, thus providing a new way for cell transplantation therapy of degenerated intervertebral discs.