评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应
Summary
该协议演示了从小鼠眼部表面细胞同时检测活原培养物中的活性氧物种（ROS）、活细胞和死细胞。2’，7′-二氯氟乙酸酸酯，碘化钠和Hoechst染色分别用于评估ROS，死细胞和活细胞，然后成像和分析。
Abstract
由于各种环境因素，眼部表面经常磨损。暴露于UV-C辐射构成职业健康危害。在这里，我们演示了从小鼠眼部表面向UV-C辐射暴露的原发干细胞。活性氧物种（ROS）的形成是氧化应激/损伤程度的读出。在实验性体外环境中，评估氧化应激产生的死细胞百分比也至关重要。在本文中，我们将演示UV-C暴露小鼠原眼表面干细胞的2’，7′-二氯氟乙酸（DCFDA）染色及其基于DCFDA染色荧光图像的定量。DCFDA 染色直接对应于 ROS 生成。我们还通过同时染色碘化（PI）和Hoechst 3332以及DCFDA（ROS阳性）和PI阳性细胞的百分比，证明了死细胞和活细胞的定量。
Introduction
眼部表面（OS）是一个功能单元，主要由角膜的外层和腺上皮组成，角膜、乳腺、美博体腺、结膜、部分眼睑边缘和内侧转导信号1。透明圆顶形角膜层将光线聚焦到视网膜上。这种血管组织由细胞成分组成，如上皮细胞、角质细胞和内皮细胞以及胶原蛋白和糖甘油2等细胞成分。该地区被眼泪排干，也提供大部分的营养。操作系统的解剖位置迫使它与外部环境直接接触，经常使其暴露于各种苛刻的成分，如明亮的光线、微生物、灰尘颗粒和化学品。此因素使操作系统容易受到身体伤害，并容易出现各种疾病。
氧化应激是由于活性氧物种（ROS）的产生与内源抗氧化防御机制3之间的不平衡引起的。ROS分为活性分子和自由基，两者都来自分子氧（O2）通过线粒体氧化磷酸化4。前者由非基类组成，如过氧化氢（H2O2）、单氧（1O2），后者包括超氧化物氧化离子（O2-）和羟基基（+OH）等物种。这些分子是正常细胞过程的副产品，它们的作用与重要的生理功能有关，如信号转导、基因表达和宿主防御5。ROS的增强生产已知产生对诸如病原体入侵，异种生物和暴露于紫外线（UV）辐射4等因素。这种ROS的过度生产导致氧化应激，导致核酸、蛋白质和脂质6等分子的损伤。
自然阳光是紫外线辐射的主要来源，由UV-A（400~320nm）、UV-B（320–290 nm）和UV-C（290~200nm）7组成。据报道，波长和光谱能量之间存在反比相关性。虽然天然UV-C辐射被大气吸收，但汞灯和焊接仪器等人工辐射会发出，因此构成职业危害。暴露于眼睛的症状包括光角炎和光角结膜炎8。ROS的产生是造成紫外线诱发细胞损伤的主要机制之一。在目前的研究中，我们演示了在暴露于UV-C的小鼠原样眼表面细胞/干细胞中使用2’，7′-二氯二氢氟乙酸二乙酸（DCFDA）染色方法检测ROS。绿色荧光是使用荧光显微镜捕获的。细胞分别与两种染料，Hoechst 33342和红色碘化钠进行反染色，以染色活细胞和死细胞。
Protocol
Representative Results
Discussion
此处描述的DCFDA染色方法使用UV-C辐射处理的小鼠原生眼部活细胞中的ROS可视化。这种染色方法的一个优点是，它还允许研究人员研究UV-C（UVC暴露后3小时）对活细胞的直接影响，以及它们同时枚举ROS阳性和死细胞百分比的枚举。此外，由于染色方法用于活细胞，细胞可以在同一介质中进一步孵育更长时间（数天），以研究UV-C辐射的延迟效应。因此，这种染色方法使得在倒置荧光显微镜下能够可视?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认Yenepoya研究中心（被认为是大学）对基础设施的支持。
Materials
| 2',7'-Dichlorofluorescein diacetate (DCFDA) | Sigma | D6883 | 2',7'-Dichlorofluorescein diacetate is fluorogenic probe and is permeable to cells. It is used for quantification of reactive oxygen species. |
| Cell culture dish (35 mm) | Eppendorf | SA 003700112 | Sterile dishes for culturing the cells. |
| DMEM High Glucose | HiMedia | AT007 | Most widely used cell culture media, contains 4500 mg/L of glucose. |
| Fetal Bovine Serum, EU Origin | HiMedia | RM99955 | One of the most important components of cell culture media. It provides growth factors, amino acids, proteins, fat-soluble vitamins such as A, D, E, and K, carbohydrates, lipids, hormones, minerals, and trace elements. |
| GlutMax | Gibco, Thermo Fisher Scientific | 35050061 | Used as a supplement and an alternative to L-glutamine. It helps in improving cell viability and growth. |
| HL-2000 Hybrilinker | UVP | Hybridization oven/UV cross linker | |
| Hoechst 33342 | Sigma | B2261 | Hoechst stain is permeable to both live and dead cells. It binds to double starnded DNA irrespective of wether the cell is dead or alive. |
| Matrigel | Corning | Basement membrane matrix | |
| MEM Non-Essential Amino Acids (100X) | Gibco, Thermo Fisher Scientific | 11140050 | Used as a supplement to increase the cell growth and viability. |
| Penicillin-Streptomycin (Pen-Strep) | Gibco, Thermo Fisher Scientific | 15140122 | Penicillin and streptomycin is used to prevent the bacterial contamination in culture. |
| Propidium Iodide | Sigma | P4170 | Fluorescent dye which is only permeable to dead cells. It binds with DNA and helps in distinguishing between live and dead cells. |
| TryplE Express | Thermo Fisher Scientific | Gentle cell dissociation agent | |
| ZOE Fluorescent Cell Imager | Bio-rad |
References
- Gipson, I. K. The ocular surface: the challenge to enable and protect vision: the Friedenwald lecture. Investigative Ophthalmology and Visual Science. 48 (10), 4391-4398 (2007).
- Sridhar, M. S. Anatomy of cornea and ocular surface. Indian Journal of Ophthalmoogy. 66 (2), 190-194 (2018).
- Betteridge, D. J. What is oxidative stress. Metabolism. 49 (2), 3-8 (2000).
- Ray, P. D., Huang, B. W., Tsuji, Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signaling. 24 (5), 981-990 (2012).
- Nita, M., Grzybowski, A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxidative Medicine and Cellular Longevity. 2016, 3164734 (2016).
- Covarrubias, L., Hernandez-Garcia, D., Schnabel, D., Salas-Vidal, E., Castro-Obregon, S. Function of reactive oxygen species during animal development: passive or active. 发育生物学. 320 (1), 1-11 (2008).
- Behar-Cohen, F., et al. Ultraviolet damage to the eye revisited: eye-sun protection factor (E-SPF(R)), a new ultraviolet protection label for eyewear. Clinical Ophthalmology. 8, 87-104 (2014).
- Izadi, M., Jonaidi-Jafari, N., Pourazizi, M., Alemzadeh-Ansari, M. H., Hoseinpourfard, M. J. Photokeratitis induced by ultraviolet radiation in travelers: A major health problem. Journal of Postgraduate Medicine. 64 (1), 40-46 (2018).
- de Jager, T. L., Cockrell, A. E., Du Plessis, S. S. Ultraviolet Light Induced Generation of Reactive Oxygen Species. Advances in Experimental Medicine and Biology. 996, 15-23 (2017).
- Degl’Innocenti, D., et al. Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity. Toxicology. 411, 110-121 (2019).
- Li, Z., et al. APC-Cdh1 Regulates Neuronal Apoptosis Through Modulating Glycolysis and Pentose-Phosphate Pathway After Oxygen-Glucose Deprivation and Reperfusion. Cellular and Molecular Neurobiology. 39, 123-135 (2019).
