Shetty Rohit, Matalia Himanshu, Nuijts Rudy, Subramani Murali, Dhamodaran Kamesh, Pandian Ramanan, Jayadev Chaitra, Das Debashish
Department of Cornea & Refractive Surgery, Narayana Nethralaya, Bangalore, Karnataka, India.
Department of Cornea & Refractive Surgery, Narayana Nethralaya, Bangalore, Karnataka, India Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
Br J Ophthalmol. 2015 Feb;99(2):272-80. doi: 10.1136/bjophthalmol-2014-305495. Epub 2014 Dec 8.
AIM/BACKGROUND: To compare the effects of accelerated corneal collagen cross-linking (ACXL) and corneal collagen cross-linking (CXL) on ex vivo-cultured limbal epithelial cells (LECs).
Day 14 cultured LECs were either unexposed (control) or exposed to different intensities of ultraviolet-A (UV-A) irradiance for different durations (3 mW for 30 min, 9 mW for 10 min, 18 mW for 5 min and 30 mW for 3 min) in the presence and absence of riboflavin. These cells were further processed for quantitative real-time PCR, vital staining, immunofluorescence staining and fluorescence-activated cell sorting (FACS) staining to evaluate the apoptotic status. Statistical analysis was performed using a Student t test.
Vital staining showed a significantly higher (p=0.004) dead cell population with 3 mW for 30 min when compared with 30 mW for 3 min exposure (p=0.225). Quantitative PCR results revealed significantly reduced abcg2 and Δnp63 mRNA levels, while FACS analysis showed an increase in ABCG2-Annexin V positive population in cells exposed to 3 mW for 30 mins. Neither reduction of mRNA expression of abcg2 and Δnp63 nor increase in FACS-stained ABCG2-Annexin V positivity was detected in cells exposed to 30 mW for 3 min. Additionally, enhanced caspase activity was detected with fluorochrome inhibitor of caspases staining and mRNA expression of caspase 3 and 9 was upregulated in cells exposed to 3 mW for 30 min, but not at 30 mW for 3 min.
The 30 mW UV-A irradiation used in ACXL appears to be safe on cultured LECs in comparison with 3 mW used in CXL.
目的/背景：比较加速角膜胶原交联（ACXL）和角膜胶原交联（CXL）对体外培养的角膜缘上皮细胞（LECs）的影响。
将培养14天的LECs分为未暴露组（对照组），以及在存在和不存在核黄素的情况下，分别暴露于不同强度的紫外线A（UV-A）辐照不同持续时间的组（3毫瓦照射30分钟、9毫瓦照射10分钟、18毫瓦照射5分钟和30毫瓦照射3分钟）。对这些细胞进一步进行定量实时PCR、活细胞染色、免疫荧光染色和荧光激活细胞分选（FACS）染色，以评估细胞凋亡状态。采用学生t检验进行统计分析。
活细胞染色显示，与30毫瓦照射3分钟（p = 0.225）相比，3毫瓦照射30分钟时死细胞数量显著更高（p = 0.004）。定量PCR结果显示，abcg2和Δnp63 mRNA水平显著降低，而FACS分析显示，暴露于3毫瓦照射30分钟的细胞中ABCG2-膜联蛋白V阳性群体增加。在暴露于30毫瓦照射3分钟的细胞中，未检测到abcg2和Δnp63 mRNA表达的降低，也未检测到FACS染色的ABCG2-膜联蛋白V阳性率的增加。此外，在暴露于3毫瓦照射30分钟的细胞中，通过半胱天冬酶荧光抑制剂染色检测到半胱天冬酶活性增强，半胱天冬酶3和9的mRNA表达上调，但在30毫瓦照射3分钟时未出现这种情况。
与CXL中使用的3毫瓦相比，ACXL中使用的30毫瓦UV-A照射对培养的LECs似乎是安全的。
