【作者】 杨晨；

【导师】 王秀丽；

【作者基本信息】 扬州大学 ， 皮肤病与性病学， 2019， 硕士

【摘要】 背景痤疮丙酸杆菌(Cutibacteriumacnes,C.acnes)的感染是促进痤疮发生发展的重要因素之一。针对C.acnes的感染,以抗生素和维A酸为基础的药物已作为重度痤疮痤疮的一线治疗方法,但是随着细菌耐药性的增加及维A酸类药物的相关不良反应,临床上需要新的更安全有效的治疗手段。5-氨基酮戊酸光动力疗法(5-aminolevulinic acid photodynamictherapy,ALA-PDT)是一种新兴的药械结合治疗方法,在多个医学学科领域得到广泛应用,尤其是中重度痤疮的治疗,然而,ALA-PDT治疗痤疮的作用机制尚未十分明确。本研究旨在明确ALA-PDT对C.acnes的体外抑制作用及对兔耳痤疮模型皮损组织中IL-17表达水平的影响。目的1.明确ALA孵育C.acnes最适药物浓度、敷药时长,及不同照光剂量ALA-PDT对C.acnes的抗菌效应,建立ALA-PDT治疗细菌感染的最佳参数;2.建立兔耳痤疮模型,探讨ALA-PDT对兔耳痤疮模型皮损治疗效果和ALA-PDT治疗前后皮损组织中IL-17表达水平及其调控机制。方法1.明确PpIX荧光强度与药物浓度和敷药时间的关系实验分为对照组和实验组,对照组为C.acnes菌悬液,实验组选用不同浓度ALA(0.0625 mg/mL、0.125 mg/mL、0.25 mg/mL、0.5 mg/mL、1 mg/mL、2 mg/mL、4 mg/mL)孵育C.acnes菌悬液,各组均于37℃恒温培养箱中孵育0h、1h、2h、4h、8h、12h、24h、48h、72h、96h,多功能酶标仪分别检测各时间点PpIX荧光强度,检测后实验组样品分别接种于BHI固体培养基表面培养并进行菌落计数。通过检测不同浓度ALA作用于细菌不同孵育时间后的PpIX荧光强度,确定ALA-PDT对C.acnes抗菌效应的最佳实验参数(光敏剂ALA浓度和敷药时长)。2.确定不同照光剂量ALA-PDT处理C.acnes的抗菌效应根据第一步实验结果选择最佳ALA浓度及孵育时间,将实验分为四组:第一组为空白对照;第二组为单药组;第三组为单光组;第四组为ALA-PDT组。第三组和第四组分别予以不同剂量(0、20、40、80、100J/cm2)的红光(632±7 nm)照射,第一组、第二组不予照光,各组样本分别通过菌落计数、活死染色、透射电镜评价不同照光剂量ALA-PDT对C.acnes的体外抑制效应。3.建立兔耳痤疮模型选用新西兰家兔,于兔耳内侧面皮内注射新鲜配制的C.acnes菌悬液(3×108个/mL),隔日进行,共计21天,诱导痤疮样皮损,为进一步研究ALA-PDT对痤疮动物模型皮损及其炎症因子的影响提供研究模型。4.检测ALA-PDT治疗前后兔耳痤疮模型皮损的改变和组织中IL-17的表达水平,并初步探讨ALA-PDT影响痤疮皮损中炎症因子变化的机制研究通过肉眼观察、组织病理学评价建模结果,将建模完成后的家兔随机分为两组,分别为ALA-PDT组和无干预组,通过观察治疗前和治疗后4h、24 h、4 d、7 d皮损大体表现及组织病理改变评价痤疮炎症变化趋势。同时,采用Real-time PCR检测ALA-PDT治疗前和治疗后4h、24h、4d、7dIL-17的表达及其上游关键转录因子RORα和RORγt的表达水平。结果1.PpIX荧光强度与药物浓度和敷药时间的关系实验组孵育0h、1h、2h、4h、8h、12h荧光强度无明显改变,12h、24h荧光强度轻微升高,48h后荧光强度明显升高;对照组荧光强度随着孵育时间延长,荧光强度未见明显改变,。因此选择48h为ALA孵育C.acnes适宜孵育时间。同时,不同浓度ALA孵育C.acnes 48h,ALA浓度为0.5mg/mL时荧光强度高于其他浓度组(p<0.05)。2.不同红光剂量ALA-PDT对C.acnes的抗菌效应菌落形成单位(Colony-formingunits,CFU)计数结果显示,单光组20J/cm2、40 J/cm2、60 J/cm2对C.acnes无明显抑制作用,当照光剂量达到80 J/cm2、100J/cm2时,C.acnes存活数量明显减少(p<0.05)。ALA-PDT组照光剂量为20-100 J/cm2各剂量均明显抑制C.acnes数量,且随着照光剂量增加,ALA-PDT杀菌效应越明显(pp<0.01)。细菌活死染色示ALA-PDT随着照光剂量增加,C.acnes存活率逐渐降低。电镜结果显示,ALA-PDT处理后,细菌的细胞壁、细胞膜及细胞质结构受损。3.兔耳痤疮模型的建立兔耳皮内注射C.acnes菌悬液可诱导痤疮样皮损,可见注射区域出现丘疹、毛囊口扩大伴脂栓、部分脓肿,能较好模拟痤疮样的皮损,为进一步研究ALA-PDT治疗痤疮及对炎症因子的影响提供了动物模型。4.ALA-PDT对兔耳痤疮模型皮损治疗效果和治疗前后组织中IL-17表达水平及初步机制皮损大体表现,ALA-PDT组在治疗后4h、24h皮损区炎症较治疗前有所加重;治疗后4d组织淋巴细胞浸润较4h、24h减轻;治疗后7d皮损好转明显且优于自身治疗前及无干预组。Real-time PCR研究结果发现ALA-PDT组IL-17、RORcα、RORγt表达水平在治疗后4h逐渐升高(p=0.0819;pp=0.0296;p=0.0426),24h后进一步升高(p=0.0018;p=0.0006;p=0.0027),治疗后 4d 表达水平较前降低(p=0.2066;p=0.0286;p=0.1945),治疗后7d表达水平较治疗前显著降低(p=0.0224;p=0.0027;p=0.0233)。结论1.C.acnes能吸收ALA并转化为PpIX,ALA-PDT体外对C.acnes有明显抑制作用,当ALA浓度为0.5mg/mL、孵育48h,随着照光剂量加大,抑菌效果越强。ALA-PDT抑菌可能通过破坏菌体结构发挥作用。2.ALA-PDT体外抑菌参数与临床治疗痤疮参数(ALA浓度5%,敷药3 h)差别迥异,推测临床ALA-PDT治疗痤疮可能不是通过直接杀灭C.acnes发挥疗效。3.C.acnes菌悬液可诱导兔耳痤疮模型,皮损可模拟临床痤疮皮损,可作为ALA-PDT治疗痤疮机制研究模型。4.ALA-PDT对痤疮皮损有明显改善作用,早期通过上调IL-17放大炎症反应,随后下调IL-17,最终有效治疗痤疮,这一调控作用可能通过两个关键转录因子RORα和RORγt实现。更多还原

【Abstract】 BackgroundCutibacterium acnes(C.acnes)infection is one of the important factors that promote the development and progress of acne.To date,antibiotics and retinoic acid have been used as a fisrt-line treatment for severe acne.However,with the increase of bacterial resistance and tretinoid-related adverse reactions,new and safer therapeutic strategy is needed in clinical practice.5-aminolevulinic acid photodynamic therapy(ALA-PDT)is an emerging tool combing the drug and devices that has been widely used in many medical disciplines,especially for the treatment of moderate to severe acne.However,the mechanism of its therapeutic effect on acne has not been very clarified.The aim of this study was to determine the inhibitory effect of ALA-PDT on C.acnes in vitro and explore the regulatory effect on the expression of IL-17 in the lesions of rabbit ear acne modelObjectives1.To determine the optimal photosensitizer concentration and incubation time of ALA with C.acnes,and the antibacterial effect of different ALA-PDT illunination doses on C.acnes,establishing the best parameters for ALA-PDT treatment of bacterial infection;2.To investigate the therapeutic effect of ALA-PDT on the acne and the expression change of IL-17 in the lesions before and after ALA-PDT treatment by developing a rabbit ear acne model Methods1.Determine the relationship between PpIX fluorescence intensity and photosensitizer concentration and incubation durationThe experiment was divided into control group and experimental group.The control group was C.acnes suspension.In the experimental group,C.acnes suspension was incubated with ALA at different concentrations(0.0625 mg/mL,0.125 mg/mL,0.25 mg/mL,0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL)and each group was incubatedat 37℃ for 0h,1h,2h,4h,8h,12h,24h,48h,72h,96h respectively.After incubation,the Multi-function microplate reader was used to detect the fluorescence intensity of PpIX at each time point.The samples after treatment in the experimental group were cultured on the BHI solid medium.The optimal experimental therapeutic parameters(the photosensitizer ALA concentration and duration of application)of ALA-PDT on C.acnes were determined by measuring the fluorescence intensity of PpIX.2.Determine the antibacterial effect on C.acnes of different illumination doses of ALA-PDT Based on the results from the first step,the optimal ALA concentration and incubation time were determined.The experiment in this step was divided into four groups:blank control,single drug group,single light group,and ALA-PDT group.The third fourth group were irradiated with red light(632±7 nm)at different doses(0,20,40,80,100 J/cm2)5 and the first second group were absence of illumination.The inhibitory effects in vitro on C.acnes of ALA-PDT with different illumination doses were evaluated by colony-forming units(CFU),live death staining and transmission electron microscopy.3.Establish a rabbit ear acne modelNew Zealand rabbits were selected in this step.Freshly prepared C.acnes bacteria solution(3×108 cells/mL)was intradermal injected into the inner side of rabbit ears every other day for 21 days to induce acne-like lesions and ultimately establishing a rabbit ear acne model to facilitate.4.To detect the changes of skin lesions and the expression of IL-17 in rabbit ear acne model before and after ALA-PDT treatment,and to explore the mechanism of ALA-PDT influencing the IL-17 expression in acne lesionsVisual observation and histopathological evaluation were used to evaluate the effect of model construction,and the developed rabbit ear acne model were randomly divided into two group of ALA-PDT treatment group and non-intervention group.The inflammation in acne lesions was evaluated by observing the general performance and histopathological changes before and after treatment at 4h,24h,4d,and 7d.At the same time,Real-time PCR was used to detect the expression of IL-17 and the two upstream key transcription factors RORa and RORγt before and after treatment with ALA-PDT at 4h,24h.4d and 7d.Results1.Relationship between PpIX fluorescence intensity and drug concentration and incubation durationThe fluorescence intensity in the experimental group showed no significant change from 0h,1 h,2h,4h,8h,to 12h,but increased slightly at 12h and 24h,and the fluorescence intensity increased significantly after 48h.Therefore,48h was identified as the optimal incubation time.In the control group,the fluorescence intensity in the C.acnes suspension group increased with the incubation time but with no significance.At the same time,C.acnes was incubated for 48 h at different concentrations of ALA.The fluorescence intensity was higher than that of other concentrations(p<0.05)when the ALA concentration was 0.5 mg/mL.2.Antibacterial effect of different red light doses of ALA-PDT on C.acnesThe results of CFU showed that the single-light with doses of 20 J/cm2,40 J/cm2,and 60 J/cm2 had no significant inhibitory effect on C.acnes.However when the irradiation dose reached 80 J/cm2,the number of survived C.acnes survival reduced significantly(p<0.05).In the ALA-PDT group,the C.acnes was significantly inhibited at a doses from 20-100 J/cm2 the bactericidal effect was significantly correalated with the increase of illumination dose(p<0.01).Live death staining showed that the survival rate of C.acnes gradually decreased with the increase of the dose of ALA-PDT.Transmission electron microscopy showed that the cell wall,cell membrane and cytoplasm of the C.acnes were damaged after ALA-PDT treatment.3.Establishment of rabbit ear acne modelIntradermal injection of C.acnes in rabbit ears can induce acne-like lesions.There are papules,enlarged hair follicles with lipid plugs and partial abscesses in the injection area.The rabbit ear acne model developed in this study simulates the acne-like skin lesions and based on this good model,further study of ALA-PDT treatment on acne and tissue inflammatory factors expression can be performed successfully.4.The effect of ALA-PDT on the lesions of rabbit ear acne model and the tissues expression change of IL-17 before and after treatment and its possible regulatory mechanismIn general,the inflammation in the lesion area aggravated gradually from 4h to 24h after treatment compared with that before treatment in the ALA-PDT group.The lymphocytic infiltration was relieved at after 4d of treatment.After 7d of treatment the lesions improved obviously and were better than that of pre-treatment and non-intervention groups.The results of Real-time PCR showed that the expression levels of IL-17,RORa and RORγt in ALA-PDT group increased after 4h of treatment(p=0.0819;p=0.0296;p=0.0426)and a further increase was observed at 24h(p=0.0018;p=0.0006;p=0.0027).After 4d of treatment,their expression started to decrease(p=0.2066;p=0.0286;p=0.1945)and reduced significantly on the 7th day after treatment(p=0.0224;p=0.0027;p=0.0233).Conclusions1.C.acnes can absorb ALA and transform it as PpIX.ALA-PDT has a significant inhibitory effect on C.acnes in vitro.When incubating with ALA(concentration:0.5mg/mL)for 48h,the antibacterial effect of ALA-PDT will enhance with the increase of irradiation dose.ALA-PDT may play a role in disrupting the structure of the bacterial.2.The in vitro antibacterial parameters of ALA-PDT were significantly different from the parameters used in clinical practise(5%ALA concentration and 3 h medication),suggesting that the curative effect of ALA-PDT on acne may not be dependent on direct eradication of C.acnes.3.Intradermal injection of C.acnes suspension can develop a rabbit ear acne model which can simulate well the clinical acne lesions and can be used as a research model for treatment effect of ALA-PDT treatment on acne.4.ALA-PDT has a significant improvement effect on acne lesions.It can up-regulate IL-17 to amplify the inflammatory response,then down-regulate IL-17,and finally effectively cure acne.This regulation effect may be achieved by two key transcription factors RORα and RORγt.更多还原