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

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

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Electro Force3200力学试验仪搭载的Bio Dynamic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。

Objective To investigate the effects of different cyclic tensile strains on the proliferation and expression ofbone marrow stromal cells(BMSCs)-cocultured human degenerated anulus fibrosus(AF)cells.Methods AF cells wereisolated from a patient with degenerated intervertebral disc degeneration(IVD),which were co-cultured with BMSCs.Thesolely cultured AF cells were used as control group.The two groups of cells were expanded in monolayer,and cyclicallystrained for3hours,which were applied0,5%,10%,15%and20%strains at a frequency of0.25Hz using BioDynamic testinstrument.A flow cytometry method was used to examine the AF cell proliferation at24hours followed the application ofcyclic tensile strains.After the total RNA was extracted,real-time PCR technology was used to detect the gene expression ofcollagenⅠand aggrecan.Results Under the same appropriate stress,the proliferative index(PI),the proportion of cells inthe period of DNA synthesis,the expression of collagenⅠand aggrecan were significantly higher in the co-cultured groupthan those of control group(P<0.05).However,the best mechanical stimulation was different in the two groups.For the AFcells,the peaks of PI,the proportion of cells in the period of S period,the expression of collagenⅠand aggrecan were foundin the10%strain group,while for the co-cultured cells,they were found in the15%strain group.Conclusion Coculturingwith 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.