| 王霖杰,李慧玲,周紫莹,等.趋化因子配体 19对人角膜上皮细胞增殖的影响[J].安徽医药,2025,29(12):2518-2522. |
| 趋化因子配体 19对人角膜上皮细胞增殖的影响 |
| Effect of C-C motif ligand 19 on the proliferation of human corneal epithelial cells |
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| DOI：10.3969/j.issn.1009-6469.2025.12.040 |
| 中文关键词: 干眼 泪液 人角膜上皮细胞 趋化因子配体 19 基质金属蛋白酶 9 |
| 英文关键词: Dry eye Tears Human corneal epithelial cells Chemokine 19 Matrix metalloproteinase-9 |
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| 中文摘要: |
| 目的研究干眼炎症反应中趋化因子配体 19(CCL19)通过调控基质金属蛋白酶 9(MMP-9)表达水平对人角膜上皮细胞(HCECs)增殖的影响。方法 2023年 3—7月体外培养 HCECs，配制 310 mOsm/L的等渗培养基和 500 mOsm/L的高渗培养基。分为等渗观察组:以等渗培养基培养 24 h，选择 50、100、200 μg/L 3个浓度的 CCL19处理 12 h；等渗对照组:以等渗培养基培养 24 h，溶剂处理 12 h；CCL19高渗观察组:以高渗培养基培养 24 h，建立干眼模型，选择 50、100、200 μg/L 3个浓度的 CCL19处理 12 h；高渗对照组:以高渗培养基培养 24 h，溶剂处理 12 h。通过倒置显微镜观察各组的细胞数目变化情况，人胆囊收缩素 /缩胆囊素八肽( CCK-8)检测各组细胞增殖情况，选取最佳浓度的 CCL19进行后续实验。采用实时荧光定量 PCR及蛋白质印迹法检测最佳浓度 CCL19作用后的 HCECs中磷脂酰肌醇 3激酶( PI3K)、蛋白激酶( AKT)及 MMP-9的表达水平与对照组比较是否差异有统计学意义，并通过对比这些差异推断其在干眼中对炎症反应以及角膜上皮细胞增殖的影响。结果显微镜及 CCK-8检测发现在等渗环境下， CCL19对 HCECs的活率具有微小的影响(等渗对照组平均细胞活率为 100%，暴露于 50、100、200 μg/ L CCL19等渗培养基的 HCECs平均细胞活率分别为 97.92%、97.94%、96.74%)，而在高渗环境下， CCL19对 HCECs的活率影响较为明显(高渗对照组平均细胞活率为 25.14%，暴露于 50、100、200 μg/L CCL19高渗培养基的 HCECs平均细胞活率分别为 20.69%、19.67%、16.16%，有明显的下降趋势)CCL19的浓度越高，对 HCECs的活率抑制作用越明显。因此我们选择高渗环境下 200 μg/L浓度的 CCL19处理 HCECs后与高渗，对照组进行实时荧光定量 PCR及蛋白质印迹法检测；实时荧光定量 PCR及蛋白质印迹法结果显示， CCL19观察组中 PI3K、AKT、MMP-9的 mRNA表达水平和蛋白表达水平与高渗对照组相比，均有显著增加( P<0.05)。结论高渗环境下， CCL19浓度水平的增高，能够促进 HCECs中 MMP-9的表达，加重炎症反应，抑制角膜上皮细胞的增殖。 |
| 英文摘要: |
| Objective To investigate the effect of C-C motif chemokine ligand 19 (CCL19) on the proliferation of human corneal epi-thelial cells (HCECs) by regulating the expression of matrix metalloproteinase-9 (MMP-9). Methods From March to July 2023,HCECs were cultured in vitro, and 310 mOsm/L isotonic medium and 500 mOsm/L hypertonic medium were prepared. They were divid-ed into an isotonic observation group, cultured in isotonic medium for 24 hours and treated with 50, 100, and 200 μg/L CCL19 for 12hours, an isotonic control group, cultured in isotonic medium for 24 hours and treated with solvent for 12 hours, a CCL19 hypertonic ob-servation group, cultured in hypertonic medium for 24 hours for the establishment of a dry eye model and treated with 50, 100, and 200μg/L CCL19 for 12 hours, and a hypertonic control group, cultured in hypertonic medium for 24 hours and treated with solvent for 12hours. The number of cells in each group was observed by inverted microscope, and human cholecystokinin octapeptide (CCK-8) wasused to detect cell proliferation in each group, and the optimal concentration of CCL19 was selected for subsequent experiments. Theexpression levels of phosphatidylinositol 3 kinase (PI3K), protein kinase (Akt) and matrix metalloproteinase 9 (MMP-9) in HCECs treat-ed with the optimal concentration of CCL19 were detected by real-time fluorescent quantitative PCR (qPCR) and Western blotting, andcompared with those in the control group to see if there were statistically significant differences. The effects of CCL19 on inflammatoryreaction and corneal epithelial cell proliferation in dry eyes were inferred by comparing these differences. Results The microscope and CCK-8 detection showed that CCL19 had a slight effect on the viability of HCECs in the isotonic environment (the average cell via-bility of the isotonic control group was 100%, and the average cell viabilities of HCECs exposed to 50, 100, and 200 μg/L CCL19 iso-tonic media were 97.92%, 97.94%, and 96.74%, respectively). In the hypertonic environment, the effect of CCL19 on the viability ofHCECs was more obvious (the average cell viability of the hypertonic control group was 25.14%, and the average cell viabilities ofHCECs exposed to 50, 100, and 200 μg/L CCL19 hypertonic media were 20.69%, 19.67%, and 16.16%, respectively, showing an obvi-ous downward trend). The higher the concentration of CCL19, the more obvious the inhibition effect on the viability of HCECs. There-fore, we selected 200 μg/L CCL19 in hypertonic environment to treat HCECs, and then compared with the hypertonic control group forqPCR and western blotting detection. The detection results showed that the mRNA and protein expression levels of PI3K, Akt andMMP-9 in CCL19 observation group were significantly higher than those in hypertonic control group (P<0.05). Conclusion The in-crease of CCL19 concentration in hypertonic environment can promote the expression of MMP-9 in HCECs, aggravate the inflammatory reaction and inhibit the proliferation of corneal epithelial cells. |
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