摘要
目的:探讨细胞内Zn2+的浓度在低氧调节髓核细胞表达金属蛋白酶(metalloproteinases,MMPs)和细胞外基质(ECM)中的作用及其机制。方法:从SD大鼠中提取髓核细胞后首先进行平面培养,然后用藻酸钠凝胶进行三维培养。采用FluoZin-3 AM染色法检测细胞内Zn2+的浓度(intracellular Zn2+concentration,[Zn2+]i);采用阿利新蓝染色分析细胞分泌蛋白多糖的含量,采用二甲基亚甲蓝分光光度法(DMMB)检测糖胺聚糖的表达水平,采用real-time PCR分析II型胶原(α1 type II collagen,COL2A1)、金属蛋白酶13(matrix metalloproteinase 13,MMP-13)和解整合素样金属蛋白酶5(a disintegrin and metalloproteinase with a thrombospondin motif 5,ADAMTS-5)mRNA的表达。通过免疫组化法和Western印迹法分析锌铁转运蛋白8(ZRT,IRT-like protein8,ZIP8)的表达水平。结果:IL-1β和ZnCl2可以显著提高髓核细胞内的[Zn2+]i,但是该作用可以被低氧所抑制。在藻酸钠三维培养的髓核细胞中,低氧可以显著改善IL-1β和ZnCl2引起的蛋白多糖、糖胺聚糖和COL2A1 mRNA的降低,但ZnCl2可以抑制低氧的保护作用。细胞内Zn2+的螯合剂和低氧可以抑制MMP-13 mRNA水平的升高。IL-1β和ZnCl2促进髓核细胞中ZIP8的表达升高,但是低氧可抑制ZIP8的表达。结论:低氧可以调节髓核细胞中Zn2+的内流,Zn2+介导了低氧对髓核细胞中MMP-13和ECM的调节作用。[Zn2+]i的变化可能参与了椎间盘退变的过程。
Objective：To explore the effect and mechanism of intracellular Zn2＋concentration（[Zn2＋]i）in hypoxia-induced regulation of metalloproteinases（MMPs）and extracellular matrix（ECM）expression in nucleus pulposus（NP）cells.Methods：NP cells from SD rats received plate culture at first and then three-dimensional culture with sodium alginate gel.[Zn2＋]i was assayed by FluoZin-3 AM staining.Proteoglycan was assayed by Alcian blue staining.Glycosaminoglycan was detected by1,9-dimethylmethylene blue（DMMB）assay.And real-time PCR were used to assay the mRNA expression ofα1 type II collagen（COL2A1）,matrix metalloproteinase 13（MMP-13）and a disintegrin and metalloproteinase with a thrombospondin motif 5（ADAMTS-5）.The expression of ZRT,IRT-like protein8（ZIP8）was assayed by immunohistochemistry and Western blotting.Results：Interleukin（IL）-1βand ZnCl2 could significantly increase the [Zn2＋]i of NP cells,however,the effect could be inhibited by hypoxia.Hypoxia did significantly attenuate the decrease of proteoglycan,glycosaminoglycan,and COL2A1 mRNA,which was induced by IL-1βand ZnCl2 treatment,in sodium alginate three-dimensional culture.However,ZnCl2 inhibited the protective effect of hypoxia.Both an intracellular Zn2＋chelator and hypoxia could inhibit the increase of MMP-13 mRNA expression.IL-1βand ZnCl2 treatment promoted the increase of ZIP8 expression in NP cells,however,hypoxia inhibited ZIP8 expression.Conclusions：Hypoxia may regulate the Zn2＋influx in NP cells.Zn2＋mediates the regulation effect of hypoxia on ECM and MMP-13.Perhaps the changes of[Zn2＋]i are involved in the process of intervertebral disc degeneration.
出处
《中国临床医学》
2015年第5期607-612,共6页
Chinese Journal of Clinical Medicine
关键词
锌
低氧
髓核细胞
代谢
Zinc
Hypoxia
Nucleus pulposus cells
Metabolism
