【作者】 黄小晏；

【导师】 杨慧兰；

【作者基本信息】 南方医科大学 ， 皮肤病与性病， 2008， 硕士

【摘要】 背景:白癜风是一种原发性的、局限性或泛发性的皮肤色素脱失症。据估计,人群中至少有1-2%人患白癜风。组织病理发现:黑素细胞部分或完全缺失,表皮基底层、真皮浅层T淋巴细胞浸润。黑素细胞消失的潜在机制尚不清楚,有3种可能机制诱导白癜风的发生:自身免疫、神经体液因素、自身细胞毒性。由于白癜风发病机制不明,因此其治疗效果不能使人满意,目前的治疗包括:局部用皮质类故醇激素、光线疗法（NB-UVB,UVB,UVA）、光化学疗法（UVB+补骨脂素）、黑素细胞移植及近两年出现的308nm准分子激光。据文献报道,不管采用何种治疗方法,白癜风患者皮损区色素恢复最主要的原因是毛囊乳头处的黑素细胞移行到表皮内增殖分化,合成黑素,并转运其至周围的角质形成细胞。308nm准分子激光属于UVB紫外光（波长位于308nm±1nm）,被推测具有和UVA、UVB相似的生物学效应和临床效果。308nm准分子激光在临床上一经用于白癜风的治疗,就显示其疗效好、见效快的优点。进两年来已有多个国家关于308-nm准分子在治疗白癜风有效性、安全性的临床研究报道。308nm准分子激光治疗机制尚不明确,推测可能有以下几个方面:（1）刺激黑素细胞增生,促进黑素生成。（2）诱导皮损处病理性T淋巴细胞凋亡。因此,本课题根据以卜这两点推测分别进行探讨。1.308nm准分子激光对角质形成细胞分泌细胞因子的影响研究依据:①既往研究表明中波紫外线（UVB）可诱导角质形成细胞（Humankeratinocyte,HKC）分泌多种细胞因子,如干细胞生长因子（SCF）、成纤维细胞生长因子（basic fibroblast growth factor,bFGF）、神经生长因子（nerve growth factor,NGF）、内皮素（Endothelin-1,ET）、集落细胞生长因子（granulocyte-macrophagecolony-stimulating,GM-CSF）等,而这些细胞因子能产生某些生物学效应,如诱导黑素细胞移行、增殖、分化及促进黑素合成。②308nm准分子激光的照射能量主要集中于表皮,只有大约20%的射线能到达真皮,即大部分能量集中于表皮。基于以上两点,因此提出假设:308nm准分子激光刺激角质形成细胞（KC）合成分泌细胞因子（SCF、GM-CSF、bFGF）,而这些角质形成细胞源性的细胞因子（部分KC源性的细胞因子已被证实）刺激无色素毛囊黑素细胞的移行、增殖、分化和黑素形成。基于这一假设,本课题拟研究308nm准分子对角质形成细胞的作用。目的:探讨308nm准分子激光对角质形成细胞（HKC）增殖及分泌细胞因子的影响。方法:用CCK-8法检测不同剂量308nm准分子激光照射人角质形成细胞（HKC）后12及24小时对细胞增殖的影响;不同剂量的308nm准分子激光照射HKC后12及24小时ELISA检测角质形成细胞培养上清液中的干细胞生长因子（SCF）、集落细胞生长因子（GM-CSF）、成纤维细胞生长因子（bFGF）的浓度变化。结果:角质形成细胞活力与照射剂量呈负相关,不同剂量照射对角质形成细胞的活力影响差异有显著性意义（F=30.672,P=0.000）,3个不同时间点间差异有显著性意义（F=206.134,P=0.000）。不同照射剂量对角质形成细胞分泌SCF的影响差异有显著性意义（P=0.014,F=6.033）,5个不同时间点间差异无显著性（F=0.966,P=0.054）;不同照射剂量对角质形成细胞分泌GM-CSF的影响差异无显著性意义（P=0.225,F=2.249）。5个不同时间点间差异无显著性（F=7.294,P=0.052）;不同照射剂量对角质形成细胞分泌bFGF的影响差异有显著性意义（P=0.018,F=11.889）,5个不同时间点间差异无显著性（F=49.105,P=0.001）。2.308nm准分子激光诱导T淋巴细胞凋亡的作用研究研究依据:①既往研究发现中波紫外线（UVB）能诱导人T淋巴细胞调亡。②T淋巴细胞是308nm准分子激光治疗的靶细胞。③局部转移生长因子（TGF-β₁）是一种表皮细胞增殖的负调节TGF-β₁能诱导多种细胞发生凋亡。UVB照射后,TGF-β₁水平升高,TGF-β₁具有免疫调节作用,提示UVB诱导局部TGF-β₁水平升高,从而抑制了局部免疫和炎症反应,促进局部色素恢复。目的:探讨308nm准分子激光对T淋巴细胞的直接和间接诱导凋亡作用及转化生长因子β₁（TGF-β₁）在诱导T淋巴细胞凋亡中的作用。方法:ELISA检测308nm准分子激光照射KC前及照射后12、24及48h HKC分泌TGF-β₁的浓度变化;308nm准分子激光照射体外分离的活动期白癜风患者外周血T淋巴细胞,流式细胞仪检测照射后48小时T淋巴细胞凋亡情况;建立transwell细胞共培养系统（角质形成细胞位于上室,T淋巴细胞位于下室）,照射前加入不同浓度的抗TGF-β₁抗体（25、250、2500pg/ml）,308nm准分子激光照射transwell上室,照射后48小时检测T淋巴细胞凋亡。结果:流式细胞术结果显示:308nm准分子激光可直接诱导T淋巴细胞凋亡;308nm准分子激光照射transwell上室可间接诱导T淋巴细胞凋亡。308nm准分子激光诱导角质形成细胞分泌TGF-β₁的增加呈剂量依赖性;照射前加入抗TGF-β₁抗体,可抑制准分子激光的诱导凋亡作用。3.结论:①308nm通过照射表皮的角质形成细胞,使角质形成细胞分泌SCF、GM-CSF、bFGF等具有促使黑素细胞增殖、分化及黑素合成的细胞因子,通过以上的细胞因子间接作用于黑素细胞。②308nm准分子激光直接照射可诱导T细胞凋亡,也可作用于角质形成细胞间接诱导T细胞凋亡,TGF-β₁在准分子激光诱导T淋巴细胞凋亡中起重要作用。更多还原

【Abstract】 Vitiligo is a major medical problem that can result in severe difficulties in social adjustment, which affects 1-2% of Chinese’s population. Vitiligo is characterized clinically by development of totally white macules, microscopically by complete absence of melanocytes, and medically by a not uncommon association with certain medical diseases, particularly thyroid disease.Three principal theories have been presented about the mechanism of destruction of melanocytes in vitiligo:1. The autoimmune theory holds that selected melanocytes are destroyed by certain lymphocytes that have somehow been activated.2. The neurogenic hypothesis is based on an interaction of the melanocytes and nerve cells.3. The self-destruct hypothesis suggests that melanocytes are destroyed by toxic substances formed as part of normal melanin biosynthesis.4. While the immediate mechanism for the evolving white macules involves progressive destruction of selected melanocytes by cytotoxic T cells, other genetically determined cytobiologic changes and cytokines must be involved. Because of differences in the extent and course of segmental and generalized vitiligo, the pathogenesis of these two types must be somewhat different. The approaches to the management of Vitiligo are as follows:The objective of repigmentation （Figs. 13-3 and 13-5） is the permanent return ofnormal melanin pigmentation. This may be achieved for local macules with topicalglucocorticoids or topical psoralens and UVA （long-wave ultraviolet light） and forwidespread macules with oral psoralens and UVA. Systemic photochemotherapy Formore widespread Vitiligo, oral PUVA is more practical. Narrow-band UVB, 311 nmThis is just as effective as PUVA and does not require psoralens. Repigmentation hasalso been achieved with the topical calcineurin inhibitors tacrolimus andpimecrolimus. Minigrafting may be a useful technique for refractory and stablesegmental Vitiligo macules.Recently 308-nm excimer laser has emerged as a promising therapy for the treatmentof Vitiligo. Results of previous study showed that it is effective and safe for adultpatients.308-nm excimer laser can emit mostly 311/312-nm light ,which is considered toposses the similar biological effect and clinical efficacy with UVA and UVB. Themechanism of excimer laser for Vitiligo treatment is unknown. And it is considered toto involve in several ways:l. stimulus the proliferation of melanocyte and synthesis ofmelanin.2. induce the apoptosis of T lymphocyte locating on white patch. So thestudy was divided into two parts:1. 308-nm excimer laser irradiation up-regulates keratinocyte-derived cytokineThe dosage of 308-nm excimer laser is focus on epidermis,only 20% of laser canreach dermis. So the hypothesis is proposed:308-nm ecimer laser could stimuluskeratinocyte to synthesis and secrete cytokine, which can stimulus melanocyte to .Soon the basis of the hypothesis, we study if 308-nm excimer effect keratinocyte andhow it work.Objectives The present study was designed to investigated whether 308-nm excimer laser affects keratinocyte-derived factors cytokine by keratinocyte cultured.Method Cultured keratinocyte was treated with various dosage of 308-nm excimer laser. The effects proliferation of keratinocyte by excimer laser were evaluated using CCK-8. ELISA was applied to evaluate the production of SCF,GM-CSF,bFGF in supernatant of HKC treated with excimer laser. Apoptosis was induced in HKC exposed to excimer laser.Result Proliferation of HKC was significantly reduced dosage-dependently with excimer laser. SCF,GM-CSF,bFGF secretion by HKC increased with increasing 308-nm excimer laser（ranged, 100-300 mJ/cm²） which showed statistically difference from the control group（0 mJ/cm²）. Secretion of those factor started after 6h of radiation. Exposure to excimer laser can induced apoptosis in HKC as assessed by DNA Ladder.Conclution Our study provides in vitro demonstrating that HKC creates a favourable milieu through the secretion of some cytokine for melanocyte growth and migration.2. This in vitro study addressed the questions "how does 308-nm excimer laser triggers direct and indirect apoptotic pathways of T cell.Method The effects of excimer laser on TGF-β₁ production by keratinocyte was evaluated using ELISA. Excimer laser irradiated the insert part of two-layer transwell system（T cell cultured on bottom part,keratinocyte cultured on insert part）. After 48h since irradiation,cell apoptosis of transwell system were evaluated by flow cytometry. An anti-TGF-β₁ antibody at three concentration of 25,250 and 2500ng/mL was added to transwell system before irradiation,the cell apoptosis was also detected after 48h since irradiation.Result TGF-β₁ secretion by keratinocyte increased with increasing excimer laser. The flow cytometry result showed that direct irradiation of excimer laser induced apoptosis of T cell. Apoptosis of T cell was also found in the bottom part of transwell system irradiated by excimer laser. The adding of anti-TGF-β1 antibody can suppressed the apoptosis of T cell induced by excimer laser.Conclusion 308-nm excimer laser triggers direct and indirect apoptotic pathways of T cell, TGF-β₁ play an important part in the course of T cell apoptosis induced by excimer laser.更多还原