张洪跃,周潘宇,汪 洋,许硕贵,王秀会.白细胞介素-17对体外培养髓核细胞增殖和代谢的影响[J].,2017,17(12):2218-2222 |
白细胞介素-17对体外培养髓核细胞增殖和代谢的影响 |
The Effect of IL-17 on the Proliferation and Metabolism of Nucleus Pulposus Cells Cultured in Vitro |
投稿时间:2017-01-10 修订日期:2017-02-08 |
DOI:10.13241/j.cnki.pmb.2017.12.005 |
中文关键词: 白细胞介素-17 细胞因子 炎症 椎间盘退变 |
英文关键词: Interleukin-17 Cytokine Inflammation Intervertebral disc degeneration |
基金项目:上海市卫生局科研计划项目(20134394) |
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中文摘要: |
摘要 目的:探究白细胞介素-17(interleukin-17,IL-17)对体外培养髓核细胞增殖和细胞代谢的影响。方法:髓核细胞取自经核磁共振影像确认需手术的退变椎间盘组织,建立体外培养体系。用2、5、10、15、20 ng/mL IL-17刺激髓核细胞72 h后,MTS法检测细胞增殖情况。用适当浓度IL-17刺激细胞48 h或96 h后,采用实时定量-PCR和免疫印迹方法检测基质和组织代谢相关基因的mRNA和蛋白表达。结果:IL-17刺激可以抑制体外培养髓核细胞的增殖,且15 ng/mL浓度的抑制作用最强。15 ng/mL IL-17刺激髓核细胞后,聚集蛋白聚糖(aggrecan ,ACAN)和I型胶原(type I collagen,COL1A1)mRNA表达水平显著下降(P<0.05),基质金属蛋白酶(matrix metalloproteinase-3,MMP3)、金属蛋白酶3组织抑制剂(tissue inhibitor of metalloproteinase-3,TIMP3)的mRNA表达水平显著上升(P<0.05)。COL2A1 mRNA的表达下降,MMP13、含Ⅰ型血小板结合蛋白基序的结聚蛋白样金属蛋白酶(a disintegrin like and metalloproteinase with thrombospondin type Ⅰmotifs-4,ADAMTS4)、ADAMTS5、TIMP1 mRNA的表达上升,但差异均不显著(P>0.05)。IL-17刺激48 h时,COL1A1的蛋白水平明显下降(P=0.010),而ADAMTS5的蛋白水平显著上升(P=0.005)。但刺激96h时,COL1A1的蛋白表达下降,ADAMTS5的蛋白表达上升,但无显著差异(P>0.05);COL2A1的蛋白表达水平显著下降(P=0.037)。结论:IL-17可抑制体外培养髓核细胞的增殖及代谢,在椎间盘的退变过程中可能发挥了重要的促进作用。 |
英文摘要: |
ABSTRACT Objective: To investigate the role of interleukin-17 (interleukin-17, IL-17) on proliferation and metabolism of nucleus pulposus cells cultured in vitro. Methods: The magnetic resonance imaging showed that nucleus pulposus cells were isolated from human degenerative disc tissues and cultured in vitro. Cells were cultured without or with different concentrations of IL-17. After 72 hours of stimulation by IL-17 of 2, 5, 10, 15, 20 ng/mL, the rate of proliferation inhibition was measured by MTS; Nucleus pulposus cells were cultured without or with an proper concentration of IL-17 for 48 or 96 hours. We used Real-time PCR and Western Blot method to measure the mRNA expression and protein expression levels of matrix macromolecules and tissue degradation genes. Results: It reported that the proliferation of nucleus pulposus cells cultured in vitro was inhibited with the stimulation of IL-17, while the inhibition effect of 15ng/mL IL-17 was significantly stronger. At the dose of 15 ng/mL, the stimulation of IL-17 contributed to multiple cellular responses, including increased mRNA expression of aggrecan (aggrecan, ACAN) and type I collagen (type I collagen, COL1A1) genes (P<0.05), and significantly decreased mRNA expression of tissue degradation genes, matrix metalloproteinase-3 (matrix metalloproteinase-3,MMP3)and tissue inhibitor of metalloproteinase-3 (tissue inhibitor of metalloproteinase-3, TIMP3) (P<0.05). The mRNA expression of COL2A1 and MMP13, a disintegrin like and metalloproteinase with thrombospondin type Ⅰmotifs-4 (a disintegrin like and metalloproteinase with thrombospondin type Ⅰmotifs-4, ADAMTS4), ADAMTS5, TIMP1 genes was increased, but the difference was not significant (P> 0.05). After 48 hours of stimulation by IL-17, the results of Western Blot showed that the level of COL1A1 was dramaticlly decreased (t=-2.814, P=0.010), however the peptidases (ADAMTS5) level was significantly increased (t=3.131, P=0.005).However, after 48 hours of stimulation by IL-17, the protein expression of COL1A1 decreased and the protein expression of ADAMTS5 increased, but there was no significant difference (P>0.05); COL2A1 protein expression level was significantly decreased (P=0.037). Conclusion: These findings suggest that IL-17 can inhibit the proliferation of nucleus pulposus cells in vitro and affect its metabolism, which leads to the degenerative changes of disc disease. |
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