目的:通过区分健康角膜和圆锥角膜(KC)的成纤维细胞和角膜基质细胞,检测基质金属蛋白酶(MMP)和金属蛋白酶组织抑制剂(TIMP)在角膜基质细胞中的体外表达。
方法:从健康和KC角膜中分离基质细胞(n=8)。正常的葡萄糖,含血清的细胞培养基(NGSC-培养基)用于培养健康人角膜成纤维细胞(HCFs)和KC人角膜成纤维细胞(KC-HCFs)。为了获得角膜细胞表型,用NGSC培养基的初始培养改为低葡萄糖,用于健康(角膜细胞)和KC细胞(KC-角膜细胞)的无血清细胞培养基。通过定量PCR和酶联免疫吸附测定(ELISA)从细胞培养上清液中测量MMP-1、-2、-3、-7、-9和TIMP-1、-2、-3的基因和蛋白表达。
结果:与HCFs相比,KC-HCFs显示MMP-2的mRNA基因表达更低。与它们各自的成纤维细胞组(HCFs或KC-HCFs)相比,角膜细胞显示TIMP-3的mRNA基因表达较高,而角膜细胞和KC-角膜细胞中TIMP-1mRNA基因表达较低。细胞培养上清液的蛋白质分析显示,与HCF相比,KC-HCF中的MMP-1浓度更低。与角质细胞相比,在KC-角膜细胞的细胞培养上清液中TIMP-1浓度较低。在HCFs和KC-HCFs中,与角质细胞和KC-角质细胞相比,MMP-1和TIMP-1的蛋白水平较高,MMP-2较低,分别。
结论:本研究表明健康细胞和患病细胞之间MMP和TIMP表达失衡。此外,MMPs和TIMPs的表达在角膜成纤维细胞和角膜细胞之间存在差异,这可能会影响体内特定的蛋白水解代谢,并有助于KC的进展。
OBJECTIVE: To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas.
METHODS: Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant.
RESULTS: KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively.
CONCLUSIONS: This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.