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Fluo‐Zin‐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 met‐alloproteinase 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 protein 8，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M M P‐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 by 1 ,9‐dimethylmethylene blue (DMMB) assay .And real‐time PCR were used to assay the mRNA expression of α1 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 protein 8(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 .
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