The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro

ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.摘要 目的：培养静止状态的人角膜基质细胞（human keratocytes），并体外评估紫外线与核黄素（riboflavin）对人角膜基质细胞的影响。 方法：实验所用细胞取自既往用于角膜移植手术后剩余的角膜巩膜环供体角膜，将其接种于添加2%胎牛血清（FBS）的DMEM/F12培养基中培养至细胞汇合。采用免疫荧光分析（immunofluorescence analysis）对培养细胞进行鉴定，检测标志物包括抗细胞角蛋白3（anti-cytokeratin-3）抗体、抗Thy-1抗体、抗α-平滑肌肌动蛋白（anti-α-smooth muscle actin）抗体及抗核心蛋白聚糖（anti-lumican）抗体。分别在紫外线或核黄素处理培养细胞前后开展免疫荧光实验。将角膜基质细胞以200 μL或500 μL胶原蛋白包被，随后加入0.1%核黄素，再以370 nm紫外线照射30分钟。培养24小时后，采用MTT比色法（MTT colorimetric assays）检测细胞毒性，通过Hoechst 33342与碘化丙啶（propidium iodide）染色评估细胞活性。 结果：细胞培养约20天即可达到汇合状态。培养细胞高表达核心蛋白聚糖，未检测到细胞角蛋白3、Thy-1及α-平滑肌肌动蛋白的表达。经交联处理后，MTT比色法结果显示细胞毒性较低；Hoechst 33342/碘化丙啶染色结果显示，所有胶原蛋白处理组的细胞凋亡率与坏死率均处于较低水平。 结论：本研究成功实现了人角膜基质细胞的体外培养，并可通过核心蛋白聚糖免疫荧光分析对其进行鉴定。MTT比色法及Hoechst 33342、碘化丙啶染色结果显示，未添加胶原蛋白的培养组细胞死亡率更高，表明胶原蛋白可保护角膜基质细胞免受紫外线的细胞毒性损伤。