中波高能紫外光诱导豚鼠皮肤色素沉着改变的实验研究

发布时间：2018-12-30 21:19

【摘要】：目的通过研究中波高能紫外光对豚鼠皮肤色素沉着的诱导作用,希望能够对其治疗白癜风的作用机制、剂量选择及安全性有初步了解,为其更好的应用于临床而提供一定的参考。方法以正常健康棕黄色豚鼠皮肤为实验模型,经背部脱毛处理后,取4个相邻近的区域依次作为对照组和低、中、高剂量组。根据最小红斑量(MED值)并参考预实验结果将照射起始剂量分别定为0、90 mJ/cm2、180 mJ/cm2、270 mJ/cm2(即0、1MED、2MED、3MED),首次照射后未发生红斑者下次照射剂量增加10-20%；发生红斑但下次照射前可恢复者维持原剂量；出现有症状的红斑、脱屑,则暂停照射致皮损缓解,且下次照射剂量减少10-20%。每周照射两次,共照射6周,每周对实验动物照射部位进行观察、拍摄,按时记录照射区皮肤色素沉着情况。全部实验结束后处死动物,分别取各组皮肤标本进行组织病理染色和免疫组织化学分析。实验分别采用肉眼评估、黑素细胞染色(Imokawa法)以及黑素颗粒染色(Masson-Fontana法)方法,研究中波高能紫外光所致色素沉着作用；应用免疫组织化学染色法对不同照射剂量组豚鼠皮肤中的一氧化氮合酶(NOS)进行检测,间接观察黑素调节因子一氧化氮(NO)的变化情况；应用组织病理常规染色方法(HE染色)观察表皮经中波高能紫外光照射后的组织学改变结果全部豚鼠均完成实验,少数豚鼠照射部位皮肤在照射初期出现红斑、脱屑等不良反应,减少剂量或暂停照射后均缓解。随着实验的进行,豚鼠照射部位皮肤逐渐对中波高能紫外光产生耐受,不良反应未再出现。各照射组均出现不同程度的色素沉着,除中、高剂量组色素沉着评分结果无显著性差异外,其余各组的色素沉着评分、平均黑素数量以及多巴阳性黑素细胞数之间的差异均具有统计学意义(P0.05),且中剂量组结果优于高剂量组；对照组无明显改变。免疫织化学染色示iNOS的表达亦呈剂量依赖式递增,各组间的差异具有统计学意义(P0.05)。HE染色结果显示各照射组表皮增生呈剂量依赖式递增,与紫外线照射正常皮肤所致的组织形态学改变相符。未观察到各剂量组中波高能紫外光照射引起的色素脱失及表皮细胞的异形性改变。结论中波高能紫外光可诱导豚鼠皮肤色素沉着,而且这种效应具有一定的剂量依赖性,但并非剂量越高越好。选择接近或略高于最小红斑量(MED)剂量作为初始剂量进行照射,可有效减少累积剂量,在取得满意疗效的同时又可降低紫外线治疗的潜在致癌风险。
[Abstract]:Objective to study the mechanism, dose selection and safety of the treatment of vitiligo by studying the inducing effect of high energy ultraviolet light on pigmentation in guinea pig skin. To provide a certain reference for its better application in clinical. Methods the normal brown guinea pig skin was used as the experimental model. After hair removal on the back, four adjacent areas were selected as control group and low, middle and high dose groups. According to the minimum erythema amount (MED) and referring to the results of pre-experiment, the initial dose of irradiation was determined to be 0 ~ 90 mJ/cm2180 mJ/cm2270 mJ/cm2 (i.e., 0 ~ 1 MED ~ (2) MED ~ (3 MED). The next irradiation dose of those without erythema was increased by 10 ~ (-20)% after the first irradiation. If erythema occurred but could recover before the next irradiation, the original dose could be maintained; if there were symptomatic erythema and detritus, the skin damage would be alleviated by pausing irradiation, and the dose of the next irradiation would be reduced by 10-20. The rats were irradiated twice a week for 6 weeks. The experimental animals were observed and photographed, and the pigmentation of the skin was recorded on time. At the end of the experiment, the animals were killed, and the skin samples of each group were stained with histopathology and analyzed by immunohistochemistry. The effects of medium wave high energy ultraviolet light on pigmentation were studied by means of naked eye evaluation, melanocyte staining (Imokawa) and melanin particle staining (Masson-Fontana). The nitric oxide synthase (NOS) in the skin of guinea pigs with different doses of irradiation was detected by immunohistochemical staining, and the changes of nitric oxide (NO) of melanin regulatory factor were observed indirectly. Histopathological routine staining (HE staining) was used to observe the histological changes of epidermis irradiated by medium wave high energy ultraviolet light in all guinea pigs. Erythema appeared in the skin of a few guinea pigs at the beginning of irradiation. Adverse reactions, such as detritus, were alleviated after reduction of dose or suspension of irradiation. With the development of the experiment, the skin of guinea pigs gradually developed tolerance to medium wave and high energy ultraviolet light, but the adverse reaction did not appear again. The pigmentation scores of each irradiation group were different, except that there was no significant difference in the pigmentation scores of the middle and high dose groups. The average melanin quantity and the number of dopa-positive melanocytes were significantly different (P0.05), and the results of middle dose group were better than that of high dose group. There was no obvious change in the control group. Immunocytochemical staining showed that the expression of iNOS was increased in a dose-dependent manner, and the differences among the groups were statistically significant (P0.05). The results of). HE staining showed that the epidermis proliferation of each irradiation group was increased in a dose-dependent manner. It is consistent with the histomorphologic changes caused by ultraviolet radiation on normal skin. The loss of pigment and the abnormal changes of epidermal cells were not observed in different dose groups. Conclusion Medium-wave high energy ultraviolet light can induce pigmentation in guinea pig skin in a dose-dependent manner, but the higher the dose is, the better. When the dose of (MED) is close to or slightly higher than the minimum amount of erythema as the initial dose, the cumulative dose can be effectively reduced, and the potential carcinogenic risk of UV treatment can be reduced while the curative effect is satisfactory.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.41

【参考文献】