【作者】 彭景；

【导师】 岑瑛；

【作者基本信息】 四川大学 ， 外科学（烧伤）（专业学位）， 2021， 硕士

【摘要】 目的:随着社会老年化趋势的加重,与年龄相关的疾病风险逐渐增加。老年人皮肤组织结构出现退行性变,并且对刺激和损伤的修复能力明显降低。皮肤鳞状细胞癌(Cutaneous squamous cell carcinoma,c SCC),是皮肤表皮细胞或其附属器细胞异型增生引起的的恶性肿瘤,其发病率随着年龄的增加而显著增加。细胞外基质(extracellular matrix,ECM)是构成恶性肿瘤细胞周围肿瘤微环境(Tumor microenvironment,TME)的重要物质基础,在肿瘤细胞的形成和进展中起着重要调节作用。通过研究年轻人皮肤组织、老年人皮肤组织及皮肤鳞癌组织间蛋白质的变化情况,寻找皮肤衰老相关蛋白与皮肤鳞癌的相关性,以期为皮肤鳞癌标志物和治疗靶标的筛选提供新的依据。材料和方法:选取四川大学华西医院2019年～2020年期间收治的鳞癌患者皮肤样本及外科患者手术中切除的多余正常皮肤组织样本共15例作为本次试验的研究对象。我们在获得患者同意后,收集了相关的临床组织样本和资料。以患者年龄及鳞癌的发生进行分组,年龄小于45岁的患者纳入年轻人组,年龄大于65岁的患者纳入老年组,年龄大于65岁且诊断鳞癌的患者纳入鳞癌组。提取15例患者皮肤正常组织及c SCC组织的石蜡样本作成切片保存,一部分行HE染色,观察年轻人皮肤组织、老年人皮肤组织及鳞癌组织在形态结构上的差异;另一部分切片通过PCT技术提取蛋白多肽,然后行DIA质谱采集技术对蛋白质进行定性与定量分析,筛选鉴定年轻人、老年人皮肤组织及皮肤鳞癌中的差异蛋白,然后对差异蛋白进行生物信息功能分析,同时针对差异比较组数据做相关性分析。结果:皮肤组织在衰老的进程中,表皮与真皮乳头层细胞减少,组织层次相对变薄,其交界面乳状突消失呈现扁平化,胶原纤维密度增加且其排列方向更加有序。与正常皮肤组织相比,鳞癌组织上皮细胞异型增生,核分裂异常伴深染,细胞拥挤排列呈癌巢状,伴局部炎症细胞浸润,可见同心圆排列的角化珠形成。我们对15个样本进行蛋白质组学分析,共鉴别到3008种特异性蛋白。老年人皮肤鳞癌与老年人正常皮肤组织相比,筛选出1001种差异蛋白;老年人皮肤鳞癌与年轻人正常皮肤相比,筛选出354种差异性表达蛋白;老年人正常皮肤与年轻人正常皮肤相比,筛选出688种差异蛋白。对三组样本比较结构进行相关性分析表明,有37种蛋白质在三组样本对比中呈共同差异性表达。通过筛查相关数据库发现,37种共同差异性表达的蛋白中有8种蛋白质来源于细胞外基质,主要包括Collagen alpha-6(VI)chain、Alpha-1-antitrypsin、Insulin-like growth factor-binding protein 7、Collagen alpha-1(II)chain、Cathepsin D、Decorin、Mimecan、Prolargin。对细胞外基质共性差异蛋白进行生物功能分析表明,主要参与细胞外基质的形态结构形成及赋予抗拉强度、与血小板源性生长因子及受体的相互作用、胶原分解代谢及细胞粘附作用等。结论:人体皮肤组织在衰老和癌变的过程中,组织形态结构和蛋白成分发生了改变。同时,衰老相关的细胞外基质蛋白通过调节细胞外基质形态结构与抗拉强度、血小板源性生长因子活性、胶原分解代谢、细胞粘附等作用机制,进而影响老年人皮肤鳞状细胞癌的形成与进展。更多还原

【Abstract】 Objective:With the aggravation of the social aging trend,the risk of age-related diseases gradually increases.The elderly skin tissue is degenerative,and the ability to repair irritation and injury was significantly reduced.Cutaneous squamous cell carcinoma(c SCC)is a malignant tumor originating from epidermal keratinocytes or skin accessory structures,and its incidence increases significantly with age.As a part of tumor microenvironment(TME),extracellular matrix(ECM)plays a crucial part in the occurrence and development of tumor cells.To research the changes of proteins in skin tissues of young people,old people and c SCC,to find out the correlation between skin aging-related proteins and c SCC,in order to provide a new basis for the screening of c SCC markers and therapeutic targets.Materials and Methods:15 samples of c SCC and redundant normal skin tissues were recruited to the research,removed from the patients admitted to West China Hospital of Sichuan University from 2019 to 2020.After the informed consent of the patient,we collected the clinical samples and information of the patients.And the samples were classified into three categories in accordance with the occurrence of c SCC and the age of the patients.Patients under 45 years old were classified as the young group,patients over 65 years old were classified as the elderly group,and patients over 65 years old and diagnosed with c SCC were classified as the c SCC group.Paraffin samples of skin tissues of all patients were extracted and cut into serial sections,and some sections were stained with HE to observe the differences in the morphology and structure of young skin tissues,elderly skin tissues and c SCC.In the other sections,protein polypeptides were extracted by Pressure cycling technolog(PCT),and the protein polypeptides were qualitatively and quantitatively analyzed by data independent mass spectrometry(Data independent acquisition,DIA).The differential proteins in the young skin tissues,old skin tissues and c SCC were screened and identified,and the bioinformatics function of the differential proteins was analyzed,then the correlation analysis was made according to the data of the difference comparison group.Results:With the aging of the skin,the epidermis and dermal nipple layer were thinner,the interface between epidermis and dermis was flatten,the density of collagen fibers increased and the orientation wsa relatively consistent.Compared with normal tissues,squamous carcinoma was characterised by significant dysplasia,abnormal mitosis with deep staining,crowded cancer nests accompanied by local inflammatory cell infiltration,and the formation of concentric keratin pearls.A total of 3008 specific proteins were identified by proteomic analysis of 15 samples.1,001 differentially expressed proteins were screened out in the elderly skin squamous cell carcinoma compared with the elderly normal skin;354 differentially expressed proteins were screened out in the elderly skin squamous cell carcinoma compared with the young normal skin;688kinds of differentially expressed proteins were screened out in the elderly normal skin compared with the young normal skin.Through the correlation analysis of the three groups,37 kinds of proteins showed common differential expression in the three comparisons.Through screening the related databases,revealed that 8 of the37 common differentially expressed proteins were derived from extracellular matrix,including Collagen alpha-6(VI)chain、Alpha-1-antitrypsin、Insulin-like growth factor-binding protein 7 、 Collagen alpha-1(II)chain 、 Cathepsin D 、Decorin、Mimecan、Prolargin.The biological function analysis of the common differentially expressed proteins of ECM revealed that they were mainly involved in the morphological formation and tensile strength of ECM,interaction with platelet-derived growth factor and receptors,collagen catabolism,cell adhesion and so on.Conclusion:In the process of aging and cancerization of human skin tissue,the morphological structure and protein composition of human skin tissues have changed.At the same time,the changes of extracellular matrix proteins related to aging affect the formation and progress of skin squamous cell carcinoma in the elderly by participating in the regulation of extracellular matrix morphology and tensile strength,platelet-derived growth factor activity,collagen catabolism process,cell adhesion and other mechanisms.更多还原