中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (45): 6706-6713.doi: 10.3969/j.issn.2095-4344.2016.45.003

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

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

武海军1，银和平2，胡继平1，刘  聪1，李树文2，白  明2，杜志才2   

  1. 1呼和浩特市第一医院，内蒙古自治区呼和浩特市  010010
    2内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市  010030
  • 修回日期:2016-10-04 出版日期:2016-11-04 发布日期:2016-11-04
  • 通讯作者: 银和平，教授，主任医师，硕士生导师，内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市 010030
  • 作者简介:武海军，男，1985年生，内蒙古自治区呼和浩特市人，汉族，2013年内蒙古医科大学毕业，硕士，主治医师，主要从事骨科创伤、脊柱方面的工作。
  • 基金资助:

    国家自然科学基金项目(gjzr12287)

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

Wu Hai-jun1, Yin He-ping2, Hu Ji-ping1, Liu Cong1, Li Shu-wen2, Bai Ming2, Du Zhi-cai2   

  1. 1First Hospital of Hohhot, Hohhot 010010, Inner Mongolia Autonomous Region, China
    2the Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • Revised:2016-10-04 Online:2016-11-04 Published:2016-11-04
  • Contact: Yin He-ping, Professor, Chief physician, Master’s supervisor, the Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • About author:Wu Hai-jun, Master, Attending physician, First Hospital of Hohhot, Hohhot 010010, Inner Mongolia Autonomous Region, China
  • Supported by:

    the National Natural Science Foundation of China, No. gjzr12287

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被引次数

3

摘要：

文章快速阅读：

文题释义：
Transwell 小室共培养实验：将Transwell小室放入培养板中，小室内称上室，培养板内称下室，将细胞A种于上室，细胞B种于下室，可以研究细胞B分泌或代谢产生的物质对细胞A的影响。Transwell共培养系统中的嵌套透明膜是经过特殊处理的，利于细胞的贴壁和生长。该系统中嵌套透明膜孔径是0.4 μm，相对分子质量大于70 000的物质不能通过，然而相对分子质量较小的如促进细胞生长的生长因子及细胞因子可以自由通过。这些特征使得Transwell共培养系统较直接共培养、三维共培养系统更容易观察细胞的生长状态，同时测定细胞代谢物质及施加干扰因素也更加方便，因此也被应用于各种细胞的共培养。
聚集蛋白聚糖：椎间盘退变始于椎间盘基质的退化，而聚集蛋白聚糖损耗则是基质退化最早的表现。椎间盘内的聚集蛋白聚糖主要分布在髓核，髓核细胞是主要分泌细胞。聚集蛋白聚糖损耗的直接后果是以髓核为主的椎间盘基质含水量减少，黏弹性下降。基质力学性能的下降会加重髓核细胞的受力劳损，而过度劳损的髓核细胞又会加速对基质的破坏，即减少有助于维持髓核黏弹性的聚集蛋白聚糖和Ⅱ型胶原的合成，增加弹性较差的Ⅰ型胶原的合成，分泌蛋白酶增多。随着胶原等蛋白质成分的改变，基质网状结构遭到破坏，髓核黏弹性进一步丧失，向纤维环均匀分散应力的作用进一步削弱。纤维环受力不均，又会引发纤维环的劳损，整个椎间盘组织的退变就此形成。

 

摘要
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。

 

 

关键词: 干细胞, 骨髓干细胞, 髓核细胞, 骨髓间充质干细胞, 非接触式共培养, 兔, 国家自然科学基金

Abstract:

BACKGROUND: Mesenchymal stem cells (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 cells.
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 cell morphology was close to that of NPCs. Additionally, the expression levels of type II collagen 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. Overall, 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 cells for the biological treatment of degenerative disc diseases. 

 

 

Key words: Bone Marrow, Mesenchymal Stem Cells, Coculture Techniques, Intervertebral Disk, Tissue Engineering

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