Rakhorst Hinne A, Tra Wendy M W, Posthumus-Van Sluijs Sandra T, Hovius Steven E R, Levendag Peter C, Kanaar Roland, Hofer Stefan O P
Department of Plastic, Reconstructive and Hand Surgery, Erasmus MC, Rotterdam, the Netherlands.
Tissue Eng. 2006 Dec;12(12):3395-403. doi: 10.1089/ten.2006.12.3395.
Oral mucositis is a major side effect of radiation therapy. Development of strategies for reduction of this problem calls for quantitative models. The goal of the present study was to test the feasibility of detecting double-strand breaks (DSBs) and DSB repair proteins upon radiation of mucosa in a 3-dimensional culture system using morphology and immunohistochemistry. Human oral keratinocytes and fibroblasts were seeded onto and into an acellular dermal carrier to produce a cultured mucosal substitute (CMS). CMSs were gamma-irradiated with 0, 2, and 12 Gy. One group received 4 Gy through 2 Gy fractions with a 24-h interval. Radiation-induced damage was quantified using hematoxylin and eosin (H&E). DSBs and DSB repair proteins were visualized and quantified using antibodies against P53 binding protein 1 (53BP1), MRE11, and RAD51. As in cell culture, CMSs showed intranuclear loci of damage and repair, mostly in the proliferative basal cell layers. Maximum percentages of damaged basal layer keratinocytes were 54.8% using H&E (12 Gy) up to 78.9% (12 Gy) for 53BP1. This study shows the feasibility of DNA repair markers to quantify radiation damage. This is an important step forward in the study of mucositis and the development of treatment and prevention strategies, proving once more the power and clinical importance of tissue engineering.
口腔黏膜炎是放射治疗的主要副作用。制定减少这一问题的策略需要定量模型。本研究的目的是在三维培养系统中，利用形态学和免疫组织化学方法，检测黏膜辐射后双链断裂（DSB）和DSB修复蛋白的可行性。将人口腔角质形成细胞和成纤维细胞接种到脱细胞真皮载体上并植入其中，以制备培养的黏膜替代物（CMS）。对CMS进行0、2和12 Gy的γ射线照射。一组以2 Gy的剂量分两次给予，间隔24小时，总剂量为4 Gy。使用苏木精和伊红（H&E）对辐射诱导的损伤进行定量。使用针对P53结合蛋白1（53BP1）、MRE11和RAD51的抗体对DSB和DSB修复蛋白进行可视化和定量分析。与细胞培养一样，CMS显示出损伤和修复的核内位点，主要位于增殖性基底层。使用H&E染色（12 Gy）时，受损基底层角质形成细胞的最大百分比为54.8%，而使用53BP1染色（12 Gy）时高达78.9%。本研究表明DNA修复标志物可用于定量辐射损伤。这是在口腔黏膜炎研究以及治疗和预防策略开发方面向前迈出的重要一步，再次证明了组织工程的强大作用和临床重要性。
