Porcine nucleus pulposus matrix induces differentiation of bone marrow mesenchymal stem cells into notochord-like cells

doi:10.3969/j.issn.2095-4344.1833

Abstract

Abstract:

BACKGROUND: As notochord cells can develop resistance to a harsh microenvironment in the intervertebral disc, it may be a suitable cell source for the treatment of degenerative diseases of the intervertebral disc. Porcine nucleus pulposus contains a large number of notochord cells throughout the life, and various cytokines in the cell matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells.
OBJECTIVE: To explore the effect of porcine nucleus pulposus matrix on the differentiation of bone marrow mesenchymal stem cells.
METHODS: Porcine bone marrow mesenchymal stem cells were isolated, cultured and identified in vitro. Porcine nucleus pulposus powder was prepared, and suspended in the DMEM complete medium to prepare the DMEM medium containing porcine nucleus pulposus matrix. Then, the bone marrow mesenchymal stem cells were induced in the prepared medium. After 3 weeks of induction, Notochord cell marker genes, including brachyury, cytokeratin 8, and cytokeratin 18, were evaluated by RT-PCR. The study protocol was approved by the Animal Ethics Committee of Jinshan Hospital affiliated to Fudan University.
RESULTS AND CONCLUSION: (1) Primary cultured bone marrow mesenchymal stem cells exhibited adherent growth. These cells had the ability of osteogenic and adipogenic differentiation under special conditions, and expressed CD90 and CD105. (2) Under the induction of porcine nucleus pulposus matrix, the cell morphology was changed, and the expression of brachyury, cytokeratin 8, and cytokeratin 18 was enhanced. To conclude, porcine nucleus pulposus matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells with notochord cell phenotype, which may provide a reliable cell source for the cell therapy of degenerative diseases of the intervertebral disc.

Key words: intervertebral disc degeneration, bone marrow mesenchymal stem cells, pig, nucleus pulposus, notochord cells, whole bone marrow culture, brachyury

CLC Number:

Li Defang, Jiang Zengxin, Zeng Qingmin, Wu Jingping, Xie Xiaocheng. Porcine nucleus pulposus matrix induces differentiation of bone marrow mesenchymal stem cells into notochord-like cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5366-5371.

Figures/Tables 2

2.1 骨髓间充质干细胞的形态最初分离的细胞呈圆形，核大，胞浆少，透明，细胞大小较为一致，呈悬浮状态；1 d后细胞开始贴壁，3 d后基本全部贴壁，且开始增殖，细胞伸展，体积增大，呈短梭形、多角形及不规则形等，但有较多杂质细胞；当细胞传代至第3代时细胞形态比较均匀，呈梭形成纤维样，彼此联系，细胞排列成漩涡状、网状或鱼群状，见图1。细胞增殖速度亦较快。 2.2 骨髓间充质干细胞分化能力鉴定结果对获得的骨髓间充质干细胞进行成骨、成脂分化诱导，以确定其是否具有干细胞多向分化潜能。成骨诱导分化3周后，细胞形态发生改变，由梭形转化为三角形、多角形等，培养基中出现黑色细小颗粒，换液时可触及培养板底磨砂感。茜素红染色显示骨髓间充质干细胞着色程度不一，细胞密集处着色较深，呈阳性反应，见图2A，B。成脂诱导分化后细胞周围分泌出许多微小油滴，随着诱导时间的延长，油滴逐渐增加并融合，且在培养基表面可见一层油滴，细胞由梭形变为类圆形，细胞内出现透亮脂滴。成脂诱导3周后进行油红O染色，显示脂滴呈红色，见图2C，D。 2.3 骨髓间充质干细胞表面标志物检测结果见图3。猪骨髓间充质干细胞表面标记物CD90、CD105免疫细胞化学染色呈阳性，DAPI染核，随机选取5个视野计数总细胞数及阳性细胞数，得出CD90阳性率为(98.0±2.1)%，CD105阳性率为(96.0±1.6)%。 2.4 猪髓核组织制备及其对骨髓间充质干细胞分化的影响 2周龄猪髓核组织呈胶冻状，黏性较强，经液氮冷冻后组织变脆，易于碾磨。为保证培养板中加入的髓核组织均衡，用DMEM完全培养基重悬，充分混匀，等量加入。3周后提取诱导分化组及未诱导组RNA，行RT-PCR检测脊索细胞标记性基因鼠短尾突变体表型(T)、细胞角蛋白8及细胞角蛋白18表达，结果显示：在β-actin作为内参情况下，诱导组3种基因的表达分别提高5.75倍(P < 0.01)，4.11倍(P < 0.01)和3.89倍(P < 0.01)，其中T基因表达水平上调尤为明显，差异有显著性意义，见图4和表2。"

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