【作者】 王雪；

【导师】 陶娟；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2021， 博士

【摘要】 第一部分影响血卟啉单甲醚光动力治疗鲜红斑痣有效性和安全性的临床因素分析背景:鲜红斑痣(Port-Wine Stain,PWS)的治疗对皮肤科医生来说是一个挑战,在大多数情况下,脉冲染料激光治疗并不能完全清除皮损。目前血管靶向的血卟啉单甲醚光动力疗法(HMME-PDT)已成为一种很有前景的治疗鲜红斑痣的替代方案。目的:分析性别、年龄、皮损类型和Fitzpatrick皮肤分型等临床因素与HMME-PDT治疗鲜红斑痣的有效性和安全性之间的相关性,为临床治疗提供一定的依据和参考。方法:回顾性分析我科2017年3月～2020年6月接受HMME-PDT的239例PWS病例资料,均随访6月。所有患者静脉泵入HMME(5 mg/kg),遮盖正常皮肤,药物输入10分钟后将皮损暴露于532 nm LED绿光下,照射功率密度80-100 m W/cm~2。每个光斑(10×10 cm~2)照射20-25分钟,由皮肤科医生根据患者治疗前与随访后经VISIA照片对比评价临床疗效,疗效按照基愈、显效、好转和无效进行评判。疼痛评分采用视觉模拟评分法(Visual Analogue Score,简称VAS):0分(无疼痛);1-3分(轻度疼痛);4-6分(中度疼痛);7-10分(重度疼痛),并记录术后的不良反应(包括厚痂、薄痂、色素沉着、色素减退、光毒性反应等)。根据患者性别、年龄、皮损类型、Fitzpatrick皮肤分型、皮损部位、皮损面积、既往有无PDL治疗、术后即刻反应进行分类,通过Pearsonχ~2test和Fisher’s exact test统计学方法分析上述临床因素对疗效、疼痛评分和不良反应的影响。结果:(1)疗效:基愈50例(20.9%),显效111例(46.4%)好转48例(20.1%),无效30例(12.6%),总有效病例数(基愈+显效+好转)为209例(87.4%);与疗效良好相关的临床因素包括女性、粉红型和紫红型皮损类型、Fitzpatrick皮肤分型Ⅲ型、皮损位于头颈部、皮损面积≤40cm~2以及术后即刻出现苍白水肿和紫癜现象(p<0.05)。(2)疼痛评分:重度97例(40.6%),中度65例(27.2%),轻度50例(20.9%),无疼痛27例(11.3%);与重度疼痛相关的临床因素包括男性、年龄5岁以上、皮损位于头颈部、皮损面积>40cm~2以及术后即刻出现颜色加深变暗(p<0.05)。(3)不良反应:出现不良反应46例(19.2%),未出现不良反应193例(80.8%);不良反应包括厚痂8例,薄痂30例,色素沉着5例,色素减退1例,光毒性反应2例,均在治疗后3-6月内恢复正常;与不良反应发生相关的临床因素包括粉红型和增厚结节型皮损类型以及既往有治疗史(p<0.05)。结论:HMME-PDT治疗鲜红斑痣是安全有效的,同时,其有效性和安全性受多种临床因素的影响,该研究结果为PWS患者的合理化和个体化治疗提供了依据,有利于实现最安全的体验和最优化的疗效。第二部分皮肤镜血管模式与血卟啉单甲醚光动力治疗鲜红斑痣疗效的关系背景:HMME-PDT是一种新颖且有效的PWS治疗策略,同时,其有效性和安全性受多种临床因素的影响,为PWS患者的合理化和个体化治疗提供了参考。但是考虑到PDT的治疗经济成本高等缺点,如何精准筛选出符合HMME-PDT治疗适应症的PWS候选人一直是临床问题,为患者提供无创皮肤镜检测,来预判疗效是很重要的,但目前关于皮肤镜的PWS血管模式预测光动力疗效的报道较少。目的:分析皮肤镜的PWS血管模式分型在预测HMME-PDT的疗效中的作用。方法:我们在2019年1月10日至2019年12月11日期间开展了一项前瞻性研究,纳入我院皮肤科门诊诊断为PWS的163例患者,均接受2次HMME-PDT,治疗间隔为两个月,治疗全部结束后随访6个月。3名皮肤科医生预先设立8种PWS皮肤镜的血管模式分型,由同一位研究人员分别在患者治疗前后采集图像,使用皮肤镜和VISIA系统记录PWS血管模式和皮损图像。由另外4名皮肤科医生通过VISIA图片评估治疗前后的疗效,并分析皮肤镜的血管模式与HMME-PDT疗效的关系。结果:皮肤镜观察PWS患者的血管模式分型发现,HMME-PDT对具有点球状(25例,15.3%)、短棒状(30例,18.4%)以及弯曲状(21例,12.9%),这三种血管模式的患者疗效最佳;在血管模式为围绕褐色点的苍白圈(13例8.0%)和树枝状(16例,9.8%)的患者中HMME-PDT治疗PWS的疗效中等;而具有混合型(21例,12.9%)、灰白幕(19例,11.7%)以及网状(18例,11.0%)这三种血管模式的患者接受HMME-PDT后疗效差或无效(p<0.05)。结论:HMME-PDT治疗PWS的疗效和皮肤镜血管模式有明确的相关性,因此在PDT治疗PWS前,皮肤镜检查可提供非常有用的临床信息。皮肤镜不仅呈现了PWS的血管模式,还有助于预测HMME-PDT的疗效,有效地管理患者的期望值。第三部分3D全层皮肤等效物疾病模型构建及药物作用机制的探索背景:临床中我们发现皮肤镜血管模式对预测疗效有意义,故在体外构建相关疾病模型,探索治疗后PWS血管损伤情况具有重要的研究价值。目前尽管动物和细胞模型为疾病提供研究平台,但物种差异性和缺乏人体皮肤复杂生理的微环境,仍然是PWS疾病研究的瓶颈问题。因此,构建一个能够模拟人体生理条件,与鲜红斑痣特性相似的体外3D模型是十分必要的。目的:基于皮肤组织工程学,在体外构建3D全层皮肤等效物的鲜红斑痣疾病模型,初步探索不同药物治疗后PWS血管损伤和作用机制。方法:将从包皮中分离出来的正常人成纤维细胞(normal human dermal fibroblasts,NHDFs)与正常人毛细血管内皮细胞(human dermal microvascular endothlial cells,HDMECs)以数量比1:1混合均匀后接种到皮肤模型支架上,连续培养28天,得到成熟的3D真皮等效物(dermal equivalent,DE)后,第一部分实验分组如下:空白对照组、PWS模型组。在第二部分实验中,我们将正常人角质形成细胞(normal human epidermal keratinocytes,NHEK)接种到成熟的3D真皮等效物上,并维持气液相培养14天,得到3D全层皮肤等效物模型(full-thickness skin equivalent,SE)后,处理分组如下:空白对照组、PWS模型组、低浓度雷帕霉素(Rapamycin,RA)组、高浓度RA组、低浓度咪喹莫特(Imiquimod,IM)组、高浓度IM组以及DMSO组。其中空白对照组仍使用常规培养基继续培养,致病因子组为加入VEGF-A、FGF、MMP-2、MMP-9(均50 ng/ml)的常规培养基连续培养7天。制作石蜡切片后进行HE染色,在显微镜下观察组织形态,用免疫组化和免疫荧光CD31等特异性染色,研究人毛细血管内皮细胞的p-PI3K、p-AKT、p-ERK和p-JNK表达,以及3D模型中类毛细血管结构的形成情况。结果:(1)DE(3D真皮等效物)免疫组化染色结果表明:与空白对照组相比,PWS模型的人毛细血管内皮细胞p-PI3K、p-AKT、p-ERK和p-JNK表达显著增强(p<0.05);DE的CD31免疫荧光染色结果表明:PWS模型组血管密度和血管直径显著高于空白对照组(p<0.05)。(2)SE(全层皮肤等效物模型)免疫组化染色评分结果表明:与空白组相比,PWS模型组的人毛细血管内皮细胞p-PI3K、p-AKT、p-ERK和p-JNK表达显著增强(p<0.05)。PWS模型组与RA处理组相比,RA 10μg/ml和RA 20μg/ml处理后p-PI3K、p-AKT表达均降低(p<0.05)其余组间未见变化(p>0.05);PWS模型组与IM处理组相比,IM 10μg/ml和IM 20μg/ml处理后人毛细血管内皮细胞p-JNK表达均降低(p<0.05),其余组间无明显差异(p>0.05)。SE(全层皮肤等效物模型)CD31免疫荧光染色结果表明:PWS模型组血管密度和血管直径高于空白对照组(p<0.05)。RA 10μg/ml和RA 20μg/ml处理组的血管密度和血管直径均明显低于PWS模型组(p<0.05),RA不同浓度组间无差异(p>0.05);IM 10μg/ml和IM 20μg/ml处理后,血管密度均明显降低(p<0.05),但IM 10μg/ml处理组的血管直径与PWS模型组无显著性差异(p>0.05),IM 20μg/ml处理组的血管直径和密度明显低于PWS模型组(p<0.05)和IM 10μg/ml(p<0.05)。DMSO处理组的血管密度和血管直径与PWS模型组相比无明显变化(p>0.05)。结论:本研究在体外成功构建了类鲜红斑痣3D真皮和全层皮肤等效物模型,该模型可作为PWS病理生理和复杂结构的研究平台,该疾病模型构建成功,可为新型药物的测试和治疗技术的筛选带来便利。更多还原

【Abstract】 PartⅠAnalysis of clinical factors affecting the effectiveness and safety of hematoporphyrin monomethyl ether photodynamic therapy of port wine stainsBACKGROUND:Port-Wine Stain(PWS)treatment is a challenge for dermatologists.In most cases,pulsed dye laser treatment cannot completely remove the skin lesions.At present,hematoporphyrin monomethyl ether photodynamic therapy(HMME-PDT)has become a promising alternative for the treatment of port wine stains.OBJECTIVE:To analyze the relationship between gender,age,skin lesion type and Fitzpatrick skin type and other clinical factors and the effectiveness and safety of HMME-PDT in port wine stains,and provide certain basis and reference for clinical treatment.METHODS:The data of 239 cases of port wine stains(PWS)treated with hematoporphyrin monomethyl ether photodynamic therapy(HMME-PDT)in our department from March 2017 to June 2020 were retrospectively analyzed.All were followed up for 6 months.All patients were pumped with HMME 5 mg/kg intravenously,the normal skin was covered,and the skin lesions were exposed to 532 nm LED green light10 minutes after drug infusion,and the irradiation power density was 80-100 m W/cm~2.Each spot(10×10cm~2)was irradiated for 20-25 minutes,according to the patient’s gender,age group,skin lesion type,Fitzpatrick skin type,skin lesion location,skin size,previous treatment history,and the type of immediate postoperative response are classified.Dermatologists evaluated the clinical efficacy based on the comparison of VISIA before and after PWS treatment.The overall efficacy were evaluated in accordance with basic recovery,marked effect,improvement,and ineffectiveness.The pain score was based on the Visual Analogue Scale/Score(VAS):0 points(no pain);1-3 points(mild pain);4-6points(moderate pain);7-10 points(severe pain),and recorded adverse reactions(thick scab,thin scab,hyperpigmentation,hypopigmentation,phototoxic reaction)after PDT.The Pearsonχ~2 test and Fischer’s exact test were used to analyzing the effects of the above factors on the efficacy,pain score,and adverse reactions.RESULTS:(1)Efficacy:50 cases of basic recovery(20.9%),111 cases of marked effect(r46.4%)improvement in 48 cases(20.1%),30 cases of ineffectiveness(12.6%),the total number of effective cases(basic recovery+marked effect+improvement)were 209 cases(87.4%);clinical factors related to good curative effect included female,pink and purple skin lesion types,Fitzpatrick skin typeⅢ,skin lesions located in the head and neck,skin lesion area≤40cm~2,and pale edema and purpura appeared immediately(p<0.05).(2)Pain score:97 cases were severe(40.6%),65 cases were moderate(27.2%),50 cases were mild(20.9%),and 27 cases were painless(11.3%);clinical factors related to severe pain including males,over 5 years of age,skin lesions located on the head and neck,skin lesions area>40cm~2,and the color became darker immediately after PDT(p<0.05).(3)Adverse reaction:46 cases(19.2%)of adverse reactions occurred,193 cases(80.8%)did not occur;adverse reactions included 8 cases of thick crust,30 cases of thin crust,5 cases of hyperpigmentation,and 1 case of hypopigmentation,and 2 cases of phototoxic reaction,which returned to normal within 3-6 months after treatment;clinical factors related to the occurrence of adverse reactions included pink type and thickened nodular skin lesion types and previous treatment history(p<0.05).CONCLUSION:HMME-PDT is safe and effective in the treatment of PWS.At the same time,its effectiveness and safety are affected by a variety of clinical factors.The results of this study provide a basis for the rationalization and individualized treatment of PWS patients,which is conducive to achieving the safest experience and optimal curative effect.Part Ⅱ The relationship between dermoscopic vascular pattern and the effect of hematoporphyrin monomethyl ether photodynamic therapy on port wine stainsBACKGROUND: HMME-PDT is a novel,safe and effective treatment strategy for PWS.At the same time,its effectiveness and safety are affected by a variety of clinical factors,which provide a basis for the rationalization and individualized treatment of PWS patients.However,considering the shortcomings of the high economic cost of PDT treatment,how to accurately screen out PWS candidates that meet the HMME-PDT treatment indications has always been a clinical problem.It is very important to provide patients with non-invasive guidance and predict the effect of curative effect.However,there are few reports about the PWS vascular model of non-invasive dermoscopy to predict curative effect of HMME-PDT.OBJECTIVE: To analyze whether the vascular pattern classifications of PWS by dermoscopy can predict the efficacy of PDT.METHODS: This prospective cohort study included 163 patients with a clinical diagnosis of PWS who were treated twice with hemoporfin-mediated photodynamic therapy(HMME-PDT)at two-month intervals and followed up for 6 months.The vascular manifestations of dermoscopy with PWS were independently classified into 8 categories by 3 dermatologists.Images of the lesions were taken using VISIA,and the vascular patterns were imaged by dermoscopy by the same investigator.Images were captured before and after each treatment.The efficacy was evaluated with pre-and post-treatment VISIA images,and correlations between efficacy and vascular patterns were analyzed by four dermatologists in a blinded and independent manner,between 10 January 2019 and 11 December 2019.RESULTS: In the dermoscopy images for the whole cohort,dotted and globular vessels (25 cases,15.3%),short clubbed vessels(30 cases,18.4%),and curved vessels(21 cases,12.9%),patients with vascular patterns had the best effect;in patients with vascular patterns of pale halos surrounding brown dots(13 cases,8.0%)and arborizing vessels(16 cases,9.8%),HMME-PDT had moderate efficacy in the treatment of PWS;Patients with mixed(21 cases,12.9%),grey-whitish veil(19 cases,11.7%),and reticular patterns(18 cases,11.0%)vascular patterns were the worst or ineffective after receiving HMME-PDT(p<0.05).CONCLUSION: There is a clear correlation between the efficacy of PDT and the dermoscopy pattern in patients with PWS.Dermoscopy may therefore provide very useful clinical information prior to treatment in these cases.In addition,the vascular manifestations of PWS determined by dermoscopy help to predict response to PDT and manage patient expectations.Part Ⅲ Construction of 3D full-thickness human skin equivalent disease model and exploration of drug action mechanismBACKGROUND: Clinically,we found that dermoscopic vascular model is of significance in predicting the efficacy,so it is of great research value to construct related disease models in vitro and explore the vascular injury of PWS after treatment.At present,although animal and cell models provide research platforms for diseases,species differences and the lack of complex physiological microenvironment of human skin remain bottlenecks in the study of PWS diseases.Therefore,it is very necessary to construct an in vitro 3D model that can simulate the physiological conditions of the human body and is similar to the characteristics of PWS.OBJECTIVE: Based on skin tissue engineering,3D full-thickness skin equivalent disease model was constructed in vitro to preliminarily explore the vascular injury and mechanism of action of PWS after different drug treatments.METHODS: The normal human dermal fibroblasts(NHDFs)and human dermal microvascular endothlial cells(HDMECs)isolated from the foreskin,were mixed evenly at a ratio of 1:1 and then inoculated into the skin model scaffold.Above,after continuous cultivation for 28 days to obtain mature 3D dermal equivalent(DE),the first part of the experiment was divided into the following groups: blank control group,PWS model group.In the second part of the experiment,we inoculated normal human epidermal keratinocytes(NHEK)into artificial dermal tissue,and maintained the full-thickness skin model for gas-liquid incubation for 14 days to obtain 3D full-thickness skin.After the full-thickness skin equivalent(SE),the treatment groups were as follows: blank control group,PWS model group,low-concentration rapamycin(Rapamycin,RA)group,high-concentration RA group,and low-concentration imiquimod(Imiquimod,IM)group,high concentration IM group and DMSO group.The blank control group was still cultured with conventional culture medium,and the pathogenic factor group was cultured with conventional culture medium supplemented with VEGF-A,FGF,MMP-2,and MMP-9(both 50 ng/ml)for 7 days.After making paraffin sections,perform HE staining,observed the tissue morphology under a microscope,and used immunohistochemistry and immunofluorescence CD31 specific staining to study the p-PI3 K,p-AKT,p-ERK and p-JNK of human capillary endothelial cells expression and the formation of capillary-like structures in the 3D model.RESULTS:(1)DE(3D dermal equivalent)immunohistochemical staining results showed that the expression of p-PI3 K,p-AKT,p-ERK and p-JNK in human capillary endothelial cells of the PWS model was significantly increased compared with the blank control group(p<0.05);The results of DE CD31 immunofluorescence staining showed that the blood vessel density and diameter of the PWS model group were significantly higher than those of the blank control group(p<0.05)(2)The results of SE(full-thickness skin equivalent model)immunohistochemical staining scoring showed that the human capillary endothelial cells of the PWS model group expressed p-PI3 K,p-AKT,p-ERK and p-JNK compared with the control group significantly enhanced(p<0.05).Compared with the RA treatment group in the PWS model group,the expressions of p-PI3 K and p-AKT were reduced after the treatment of RA 10 μg/ml and RA 20 μg/ml(p <0.05),there was no change in the rest of the groups(p>0.05);compared with the IM treatment group in the PWS model group,the expression of p-JNK in human capillary endothelial cells decreased after IM 10 μg/ml and IM 20 μg/ml(p<0.05),and there was no significant difference between the other groups(p>0.05)).The results of SE(full-thickness skin equivalent model)CD31 immunofluorescence staining showed that the blood vessel density and diameter of the PWS model group were higher than those of the blank control group(p<0.05).The blood vessel density and diameter of the RA 10 μg/ml and RA 20 μg/ml treatment groups were significantly lower than those of the PWS model group(p<0.05),and there was no difference between the groups of different RA concentrations(p>0.05);the blood vessel density of IM 10μg/ml and IM 20μg/ml group was significantly lower than that of the PWS model group(p<0.05),but the blood vessel diameter of IM 10μg/ml was not different from that of the PWS model group(p>0.05);the blood vessel diameter of IM 20μg/ml was significantly lower than that of the PWS model group(p <0.05),and the blood vessel density and diameter of different IM concentration groups were different(p <0.05).There was no difference in blood vessel density and blood vessel diameter between the DMSO treatment group and the PWS model group(p>0.05).CONCLUSION: In this study,3D dermal and full-thickness skin equivalent models were successfully constructed in vitro,which can be used as a research platform for the pathophysiology and complex structure of PWS.The successful construction of the disease model can bring convenience for the testing of new drugs and the screening of therapeutic technologies.更多还原