【作者】 王晓林；

【导师】 孙青；

【作者基本信息】 山东大学 ， 皮肤性病学（专业学位）， 2019， 博士

【摘要】 目的:光动力疗法(photodynamic therapy,PDT)近年来主要应用于肿瘤治疗领域,其原理是通过系统性或局部应用光敏剂后,给予特定波长的光源照射,使光敏剂发生一系列的光化学反应,产生单线态氧,从而破坏细胞结构及功能继而杀伤靶细胞。光动力疗法的作用机制可能包括:一促进细胞凋亡或坏死;二损伤组织的供血血管;三影响局部或全身性的特异性或非特异性免疫反应。众所周知,银屑病的发病涉及相互影响的三个方面::角质形成细胞过度增殖、异常分化、炎症持续存在,Treg/Th17介导的免疫功能紊乱。银屑病中角质形成细胞的过度增殖被认为与肿瘤细胞的无限复制潜力相近似,因此近年来有很多学者尝试光动力疗法治疗局限性的斑块型银屑病,并取得了一定的临床疗效。同时发现,光动力疗法不仅可影响角质形成细胞的增殖分化和蛋白表达,对皮损局部和系统性免疫、炎症因子、细胞因子的合成分泌也有一定影响。但光动力疗法治疗银屑病的确切机制尚未明确,这方面的基础研究仍然很少。1960年Rothberg等发现银屑病皮损处特异性高表达角蛋白17,其表达程度与疾病严重程度密切相关,因此角蛋白17被称为“银屑病相关性细胞角蛋白”,以角蛋白17为靶点治疗银屑病也得到了一定的认可。临床中银屑病的经典治疗如阿维A,阿达木单抗等均可显著下调角蛋白17的表达。HaCat细胞被称为永生化的角质形成细胞,常被用做体外的银屑病研究模型。经过干扰素-γ的诱导,HaCat细胞可在体外过度表达角蛋白17,因此本研究尝试以干扰素-γ诱导的HaCat细胞作为研究对象,探讨5 a-氨基酮戊酸(5-aminolaevulinic acid,ALA)光动力疗法对HaCat细胞增殖,凋亡和角蛋白17表达的影响,进一步探讨光动力疗法治疗银屑病的可能分子机制,为光动力疗法治疗银屑病提供理论依据。方法:1.常规培养HaCat细胞,用250U/mL的TFN-γ刺激细胞48h,Real-time PCR法和Western-Blot法检测角蛋白17的mRNA和蛋白水平;2.用0、0.1、1、10 mmol/L浓度的ALA溶液孵育HaCat细胞4h后,635nm红光(70J/cm2)照射,MTT法检测细胞增殖活性,Real-time PCR法和Wester n-Blot法检测角蛋白17的mRNA和蛋白水平;3.用1 mmol/L ALA溶液和635nm红光(70J/cm2)干预IFN-γ诱导的HaCat细胞,孵育时间均为4h,干预后6,12,24,48h后MTT法分别检测细胞增殖活性;Real-time PCR法和Western-Blot法检测角蛋白17的mRNA和蛋白水平;4.用1 mmol/L ALA溶液,635nm红光(70J/cm2)分别和共同干预IFN-γ诱导的HaCat细胞,孵育时间均为4h,流式细胞仪检测细胞凋亡比例;5.Western-Blot法检测 1 mmol/L ALA-PDT干预前后HaCat细胞caspase3和P ARP蛋白的表达;6.Western-Blot法检测 1 mmol/L ALA-PDT干预前后HaCat细胞p-p38,p-ERK,p-JNK蛋白水平的变化;7.统计学分析 结果采用SPSS19.0统计软件进行数据分析。所有计数资料均取均数±标准差,多组均数的比较采用单因素方差分析,两组数据均数比较采用t检验,p<0.05为有显著性差异。结果::1用250u/ml IFN-y诱导HaCaT细胞48h后角蛋白17吸光度值为0.37±0.03,高于空白对照组(0.09±0.02),差异具有统计学意义(p<0.05);实验组角蛋白17的mRNA水平为3.07±0.13,高于空白对照组(1.00±0.04),差异具有统计学意义(p<0.05);2 0.1mM、1mM、10mM的ALA光动力疗法处理对IFN-γ诱导HaCat细胞增殖抑制率分别为20.28%、34.87%、44.37%,且在实验范围内抑制作用与ALA浓度成正相关;3 1mM ALA光动力疗法对IFN-γ诱导的HaCat细胞增殖抑制率在处理后6、12、24、48小时后分别为28.199%、34.86%、42.44%、50.88%,抑制作用在48小时内随时间逐渐增强;4 lmM ALA光动力疗法处理后HaCat细胞角蛋白17吸光度值为0.22±0.10,显著低于空白对照(0.67±0.04),单纯加入1mM ALA(0.63±0.04),单纯LED光源照射(70J/cm2)(0.47±0.15)(p<0.05);1mM光动力疗法处理后角蛋白17mRNA水平为0.50±0.01,显著低于空白对照(1.00±0.06),单纯加入lm M ALA(0.99±0.03),单纯LED光源照射(0.65±0.03)(p<0.05);5 lmM ALA光动力疗法处理IFN-γ诱导的HaCat细胞角蛋白17吸光度值在处理后6、12、24、48小时分别为0.62±0.04、0.41±0.03、0.28±0.03、0.11士0.03,显著低于空白对照0.70±0.02(p<0.05),抑制角蛋白17表达的作用在48小时内逐渐增强;6 lmM ALA光动力疗法处理后IFN-γ诱导HaCat细胞凋亡率为47.83%,显著高于对照组11.60%%p<0.05);光动力疗法处理后HaCat细胞caspase3,PAP蛋白吸光度值分别为0.36±0.02、0.49±0.07,显著高于对照组0.12±0.01、0.21±0.03(p<0.05);7 lmM ALA光动力疗法处理后HaCat细胞p-p38(0.47±0.05)显著高于处理前(0.12±0.02)、p-JNK(1.07±0.08)显著高于处理前(0.33±0.04)(p<0.05);而光动力疗法处理后p-ERK(0.17±0.03)降低(0.57±0.09)(p<0.05)。结论:ALA光动力疗法可显著抑制IFN-γ诱导的HaCat细胞增殖,促进其凋亡,这一作用可能通过激活p38,JNK信号传导通路实现;ALA光动力疗法对IFN-y诱导的HaCat细胞角蛋白17的高表达有显著抑制作用,且该作用与浓度和时间在一定范围内呈正相关。更多还原

【Abstract】 OBJECTIVE:Photodynamic therapy has been applied in neoplasm treatment in recent years.The effects of photodynamic therapy depend on the absorption of harmless visible light after systematic or topical application of photosensitizer,which then produces reactive oxygen species(ROS),such as singlet oxygen,that destroy target cells(cancer cells,blood vessels,and pathogenic micro-organisms)or change function.The possible mechanism of photodynamic therapy includes:promoting cell apoptosis/necrosis;damaging vessels in target tissue;participating in local/systematic innate/acquired immunity.As we know keratinocytes overproliferation and differentiation abnormality,persistent inflammation and immunity disfunction mediated by Treg/Thl7 cells.In psoriasis overproliferation of keratinocytes was considered as similar as tumor cells,some researchers tried to treat localized plaque psoriasis with photodynamic therapy and obtained certain effects.They also found PDT had influences on proliferation and differentiation,protein expression in keratinocytes,local or systemic immune status,synthetise and secretion of inflammatory factors/cytokines.But the exact mechanism of photodynamic therapy in treating psoriasis stayed unclear.,the experimental research was stilled needed.In 1960 Rothberg found keratin 17 was overexpressed in psoriasis lesions specifically which related with disease severnty and progression positively.Researchers proposed the keratin 17 as index of disease severity and effects in psoriasis,so keratin 17 was named as "psoriasis relative keratinocyte keratin",and regimens targeting keratin 17 were approved.Some classic therapies for psoriasis(eg acitretin,adalimumab et al)can downregulate the expression of keratin 17 markedly.HaCat cell is human immortalized keratinocyte which was usually used as psoriasis model in vitro.Keratin 17 was the only kereatin induced by IFN-,yin vitro,so we selected HaCat cell induced by IFN-y as subject to investigate the impacts of photodynamic therapy with 5-aminolevulinic acid on K17 expression,and to discuss the possible mechanism of photodynamic therapy treating psoriasis for the purpose of theory evidence.MATERIALS AND METHODS:1.Inducion of K17 expression in HaCaT cells:HaCaT cells were stimulated with IFN-y(250 U/ml)for 48 h,after which the cells were used for proliferation assays;2.Detection on the impacts of ALA-PDT onHaCaT cells proliferation in vitro by MTT Assay:use different concentration ALA(0、0.1、land 10 mM)and same power density(70J/cm2)to intervene HaCat cells,collect cells respectively after 6h、12h、24h and 48h and use the MTT assay to detect the cells proliferation activity;3.Detection on the impacts of ALA-PDT on K17、PAPR、caspase-3、p-p38、p-ERK、p-JNK protein expression in HaCaT cells by Western Blot methods;4.Detection on the impacts of ALA-PDT on K17 mRNA expression in HaCaT cells by Real-time PCR;5.Detection on the impacts of ALA-PDT on HaCaT cells apotosis by Annexin V/7-AAD Staining;6.Statistical analysis:Then we did statistic analysis with SPSS 19.0 software.All the counting data were taken as the standard deviation of the mean.The comparison of the multigroup mean was made by single factor analysis of variance.The difference between the two groups was statistically analysed using t test,P<0.05 was significant difference.RESULTS:1.The absorbance value of keratin 17 in HaCaT cells 48 hours after induction with 250u/ml IFN-γ was 0.37±0.03,higher than that in control group(0.09±0.02)(p<0.05);the mRNA level of keratin 17 in experimental group was 3.07±0.13,higher than that in control group(1.00±0.04)(p<0.05);2.The inhibition rates of IFN-y induced HaCat cells were 20.28%,34.87%and 44.37%respectively under 0.1mM,l1mM and lOmM ALA photodynamic treatment,and the inhibition rate was positively correlated with the concentration of ALA in the experimental range;3.The inhibitory rates of 1 mM ALA photodynamic therapy on IFN-y induced HaCat cells were 28.19%,34.86%,42.44%and 50.88%at 6,12,24 and 48 hours after treatment respectively.The inhibitory effects gradually enhanced with time within 48 hours;4.The absorbance value of keratin 17 in HaCat cells treated with 1 mM ALA-PDT was 0.22 ± 0.10,which was significantly lower than that in control group(0.67±0.04).The levels of keratin 17 protein in HaCat cells treated with 1 mM ALA singly were 0.63 ± 0.04 and irradiation at 70 J/cm 2 singly(0.47±0.15)(p<0.05).After 1 mM ALA-PDT the level of keratin 17 mRNA was 0.5010.01,and lower than that in control(1.00±06)(p<0.05),only 1 mALA(0.99±0.03)and only irradiation at 70 J/cm2(0.65±0.03)(p<0.05);5.The absorbance of keratin 17 induced by IFN-γ in HaCat cells treated with ImM ALA was 0.62 ± 0.04,0.41 ± 0.03,0.28 ±0.03 and 0.11 ±0.03 respectively at 6,12,24 and 48 hours after treatment,which was significantly lower than that of control 0.7010.02(p<0.05),and the inhibitory effect enhenced gradually within 48 hours;6.The apoptosis rate of HaCat cells induced by IFN-y was 47.83%after 1 mM ALA photodynamic treatment,which was significantly higher than that of the control group 11.60%(p<0.05).The absorbance values of PARP protein and caspase 3 in HaCat cells after photodynamic treatment were 0.36 ±0.02 and 0.49 ± 0.07 respectively,which were significantly higher than those in the control group(0.12 ± 0.01 and 0.21 ±0.03)(P<0.05);7.P-p38(0.47±0.05)and p-JNK(1.07±0.08)of HaCat cells treated with 1 mM ALA PDT were significantly higher than those before treatment(0.12±0.02 and 0.33±0.04)(p<0.05),while p-ERK(0.1 7±0.03)was significantly lower than that before treatment(0.57±0.09)(p<0.05)CONCLUSIONS:ALA photodynamic therapy can significantly inhibit proliferation and promote apoptosis of IFN-y induced HaCat cells,this effect may be achieved by activating p38,JNK signal transduction pathway;ALA photodynamic therapy can significantly inhibit IFN-y induced expression of keratin 17 in HaCat cells,and the effect is positively correlated with concentration and time in a certain range.更多还原