^(32)P-β射线致急性放射性皮肤损伤模型的建立及损伤机制

Establishment of an acute radioactive skin injury model induced by ^(32)P-beta ray radiation and the mechanism of injury

背景:急性放射性皮肤损伤的临床表现为反复发作的坏死性溃疡,其发病机制仍不完全清楚,建立合适的动物模型对其发病机制及预防治疗的研究具有重要临床价值。目的:建立急性β射线放射性皮肤损伤模型,初步探究其损伤机制。方法:69只SD大鼠随机分为30,45和^(60)Gyβ射线照射组(n=21)及对照组(n=6),采用^(32)P放射性核素对大鼠背部进行单次局部照射,对照组除不照射外其余操作与各照射组相同。造模后监测各组大鼠体质量,各照射组分别于照射后第7,15,30,45,60天各取3只大鼠切取皮肤组织观察损伤情况,包括皮肤外观、苏木精-伊红染色、Masson染色、透射电镜、TUNEL实验观察,另外采用免疫组化、Western Blot观察皮肤样本中Bcl-2、Bax及P53凋亡相关蛋白的表达。结果与结论:①照射后大鼠无意外死亡,体质量呈逐渐增加趋势;②大鼠皮肤出现不同程度的表皮层坏死、炎症细胞浸润、毛囊及附属器减少、胶原纤维断裂,以^(60)Gy、^(45)Gy表现明显,血清炎症因子白细胞介素6、肿瘤坏死因子α水平显著升高,呈剂量依赖性;③电镜结果显示细胞内出现不同程度的线粒体数量减少、空泡化以及核固缩,细胞凋亡程度呈现一定的剂量依赖性;④免疫组织化学及Western Blot结果显示,照射后皮肤组织P53及Bax蛋白表达增加,Bcl-2蛋白表达减弱,^(45)Gy、^(60)Gyβ射线组与30 Gyβ射线组相比差异有显著性意义(P<0.05);^(60)Gyβ射线组与^(45)Gyβ射线组相比差异有显著性意义(P<0.05);⑤结果提示,^(32)P放射性核素^(45)Gy及^(60)Gy照射大鼠背部可成功建立急性放射性皮肤损伤动物模型,其机制与凋亡相关因子P53、Bax上调及Bcl-2下调有关,该模型可为放射性皮肤损伤预防治疗及相关机制研究提供参考。

BACKGROUND:The clinical manifestation of acute radiation skin injury is recurrent necrotic ulcers,and its pathogenesis is still not fully understood.The establishment of a suitable animal model will have important clinical implications for the study of its pathogenesis,prevention and treatment.OBJECTIVE:To establish a model of acuteβ-ray radiation skin injury and to investigate the mechanism of injury.METHODS:Sixty-nine Sprague-Dawley rats were randomly divided into 30,45,^(60)Gy ^(32)P-β-ray groups(n=21 per group)and control group(n=6).A single local irradiation of the back of the rats was performed using ^(32)P radionuclide.The control group was operated in the same way as the irradiated groups except that it was not irradiated.The body mass and skin appearance of the rats were measured at 7,15,30,45,and 60 days after irradiation.Three rats from each group were selected at each observation time point.The skin injury was observed by hematoxylin-eosin staining,Masson staining,transmission electron microscopy,and TUNEL assay.P53,Bcl-2 and Bax protein levels in the skin were measured by immunohistochemistry and western blot assay.RESULTS AND CONCLUSION:There was no accidental death after irradiation,and the body mass of rats showed a gradual increase.The rats showed different degrees of epidermal necrosis,inflammatory cell infiltration,reduction of hair follicles and appendages,and collagen fibrillation,which were evident at 60 and ^(45)Gy.The levels of serum inflammatory factors,interleukin-6 and tumor necrosis factor-α,were significantly increased in a dose-dependent manner.Under the electron microscope,there are varying degrees of mitochondrial reduction,vacuolization and nuclear pyknosis in the cells.The degree of cell apoptosis showed a certain dose-dependence.Immunohistochemistry and western blot results showed an increase in the expression of P53 and Bax proteins and a decrease in the expression of Bcl-2 protein in the skin after irradiation.There were significant differences between the ^(60)Gy group and the ^(45)Gy and 30 Gy groups(P<0.05).To conclude,irradiation with ^(60)Gy and ^(45)Gy ^(32)P radionuclide on the back of rats could successfully establish a practically pre-clinical animal model,and the mechanism is related to the up-regulation of P53 and Bax and the down-regulation of Bcl-2.This model can provide a reference for the establishment of animal models for the study of the mechanism of radiation skin injury and its prevention and treatment.