光敏剂甲苯胺蓝在口腔颊粘膜渗透性的研究

Fluorescence Biodistribution of Toluidine Blue O on Rat Buccal Mucosa

目的:研究不同时间,甲苯胺蓝(Toluidine Blue O,TBO)在大鼠炎症性口腔颊粘膜渗透的浓度变化,及甲苯胺蓝与炎症细胞分布之间的关系。方法:实验选取wistar大鼠32只,炎症组大鼠20只,建立以金葡菌为优势菌的感染炎症创口模型。将浓度为1mg/mL甲苯胺蓝溶液置于大鼠感染炎症创口组织上5、10分钟后处死,正常粘膜组大鼠8只,于甲苯胺蓝在正常颊粘膜渗透5、10、20、40分钟后处死,避光条件下取组织块进行冰冻切片,即刻荧光显微镜下观察荧光分布;冰冻切片进行HE染色,观察炎症细胞分布。采用Image Pro-plus 6.0软件检测荧光分布的光密度、分布面积以及炎症分布面积。结果:1创口周围正常粘膜及正常完整粘膜组的甲苯胺蓝均停留在角化层,未穿透上皮层,和时间无相关关系;2炎症5分钟组平均荧光分布可达到炎症细胞分布面积的89%,炎症10分钟组可达108%;炎症组在创口表面及深部,荧光光密度均无显著差异。结论:1甲苯胺蓝可有效分布于感染的炎症组织,但不能穿透正常组织,完整上皮可保护正常组织免受光动力的杀伤。2浓度为1 mg/mL的甲苯胺蓝溶液渗透时间为10分钟时,创口中甲苯胺蓝的分布与炎症细胞的分布基本一致,甲苯胺蓝浓度梯度无显著变化。提示甲苯胺蓝作为光敏剂在针对口腔创口感染的抗菌光动力疗法中可有效、安全的发挥作用。

Objective： This study focused on the timely change of osmotic concentration of toluidine blue（Toluidine Blue O,TBO） on the rat buccal mucosa, and the corresponding relationship between the distribution of toluidine blue and inflammatory cells.Methods： 32 wistar rats, were chosen and divided into three groups, namely inflammation group, control group and blank group. The 20 rats in the inflammatory group were cultured by the type strain of Staphylococcus aureus to established the wound model of inflammation. Primary TBO solution（1mg/mL） was placed on the wound model. Ten rats of the inflammation group were sacrificed after five minutes, the rest rats of the inflammation group were killed 5 minutes later. The specimens were frozen and sliced to observe the fluorescence distribution, and melt were stained with HE to observe the distribution of inflammation cells. With Image Pro-plus 6.0 software for image processing. After the experiment, parameters, such as the optical density of fluorescence distribution, the area of fluorescence distribution and distribution area of inflammation were measured. Results： 1The photosensitizer（PS） was localized in the intact epithelial layers and its distribution was not related to time. 2 In 5-minute inflammation group, the average fluorescence distribution of inflammatory cells reached 89%, and 10 minutes group could reach 108%. In the inflammatory group, no significant differences of optical density of fluorescence distribution was noticed though the wound. Conclusion： 1Toluidine blue can be effectively distributed in the infected tissue, yet can not penetrate the normal tissues. Inhance intact epithelium may protect normal tissues from Photodynamic killing. 2 TBO solution（1mg/mL） was placed on the wound model for 10 minutes, the distribution of toluidine blue consistented with the distribution of inflammatory cells, with on significant change of luidine blue in concentration gradient. These promising results suggested the use of such treatment as a safe and effective alternative to topical anti-microbials in future clinical applications.