天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (6): 571-576.doi: 10.11958/20161109

• 细胞与分子生物学 • 上一篇    下一篇

循环牵张应力下 BMSCs 非接触共培养对人退变 纤维环细胞增殖及蛋白表达的影响

李爽 1，孙晓雷 1，马信龙 1△，张杨 2，邓树才 1，郝永宏 1   

  1. 1 天津市天津医院骨科（邮编 300211），2 骨科研究所
  • 收稿日期:2016-10-09 修回日期:2017-05-02 出版日期:2017-06-15 发布日期:2017-07-05
  • 通讯作者: △通讯作者 E-mail: xlspine@163.com E-mail:maxinlong8686@sina.com
  • 作者简介:李爽（1984），男，住院医师，博士在读，主要从事脊柱外科及椎间盘修复等相关研究
  • 基金资助:
    天津市卫生局科技基金（2012KZ052）

The response of cyclic tensile strain on the BMSCs co-cultured human degenerative anulus fibrosus cells

LI Shuang1, SUN Xiao-lei1, MA Xin-long1△, ZHANG Yang2, DENG Shu-cai1, HAO Yong-hong1   

  1. 1 Department of Orthopedics, 2 Institute of Orthopedics, Tianjin Hospital, Tianjin 300211, China
  • Received:2016-10-09 Revised:2017-05-02 Published:2017-06-15 Online:2017-07-05
  • Contact: △Corresponding Author E-mail：xlspine@163.com E-mail:maxinlong8686@sina.com
  • Supported by:
    Tianjin Municipal Health Bureau of science and Technology Fund

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摘要： 目的 比较不同大小循环牵张应力对共培养状态下人退变纤维环细胞增殖与蛋白表达的影响。方法 取手术摘除的腰椎间盘突出症患者椎间盘，病理学方法对其退变程度进行分级，选取其中退变纤维环组织。酶消化 法分离纤维环细胞，选取 P3 代细胞，与术中获得的骨髓基质干细胞（BMSCs）进行非接触式共培养。利用 ElectroForce3200 力学试验仪搭载的 BioDynamic 生物反应器系统，以 3 h 为固定时间点对其进行不同大小的频率为 0.25 Hz 的周期性牵张应力刺激，设置形变量分别为 0、5%、10%、15%和 20%，同时设定无共培养的纤维环细胞为对 照组。应用流式细胞仪检测不同应力刺激下 2 种纤维环细胞的增殖情况，Real-Time PCR 方法检测人退变纤维环细 胞对蛋白多糖和Ⅰ型胶原的表达情况。结果 在相同应变刺激下，共培养组细胞的细胞增殖指数（PI）值、S 期细胞 比例、Ⅰ型胶原、蛋白多糖的表达量均高于对照组（P＜0.05）。退变组细胞 PI 值、S 期细胞比例、Ⅰ型胶原、蛋白多糖 的表达量最高值出现在 10%形变刺激下，而共培养组上述指标中除 S 期细胞比例峰值出现在 10%形变刺激下以外， 其余各指标峰值均出现在 15%形变刺激下。结论 BMSCs 的非接触共培养对人退变纤维环细胞增殖及蛋白表达存 在正向调节作用，且该调节作用受到力学环境的影响。

关键词: 椎间盘移位, 细胞, 培养的, 细胞增殖, 椎间盘退变, 循环牵张应力, 纤维环细胞, 共培养, 骨髓基质干细胞

Abstract: Objective To investigate the effects of different cyclic tensile strains on the proliferation and expression of bone marrow stromal cells (BMSCs)- cocultured human degenerated anulus fibrosus (AF) cells. Methods AF cells were isolated from a patient with degenerated intervertebral disc degeneration (IVD), which were co-cultured with BMSCs. The solely cultured AF cells were used as control group. The two groups of cells were expanded in monolayer, and cyclically strained for 3 hours, which were applied 0, 5%, 10%, 15% and 20% strains at a frequency of 0.25 Hz using BioDynamic test instrument. A flow cytometry method was used to examine the AF cell proliferation at 24 hours followed the application of cyclic tensile strains. After the total RNA was extracted, real-time PCR technology was used to detect the gene expression of collagen Ⅰ and aggrecan. Results Under the same appropriate stress, the proliferative index (PI), the proportion of cells in the period of DNA synthesis, the expression of collagen Ⅰ and aggrecan were significantly higher in the co-cultured group than those of control group (P＜0.05). However, the best mechanical stimulation was different in the two groups. For the AF cells, the peaks of PI, the proportion of cells in the period of S period, the expression of collagen Ⅰ and aggrecan were found in the 10% strain group, while for the co- cultured cells, they were found in the 15% strain group. Conclusion Coculturing with BMSCs has a positive effect on the proliferation and expression of human degenerative fibrous ring cells, which can protect AF cells from bad stress stimulation.

Key words: intervertebral disk displacement, cells, cultured, cell proliferation, intervertebral disc degeneration, cyclic tensile strain, anulus fibrosus cells, co-culture, BMSCs