【作者】 李雪；

【导师】 周良；

【作者基本信息】 南方医科大学 ， 公共卫生（专业学位）， 2021， 硕士

【摘要】 背景随着环境污染严重、日晒防护不当、日光浴的流行导致皮肤接受日照机率增加,以及人口老龄化结构等因素的影响,皮肤黑色素瘤的发病率和死亡率也逐年升高。以往的临床经验提示黑色素瘤对常规放疗不敏感。目的研究PURPL在人黑色素瘤中的生物学功能,进一步探讨PURPL在人黑色素瘤电离辐射抗性中的功能,阐明其在黑色素瘤电离辐射抗性中的作用机制,为人黑色素瘤的临床治疗提供理论依据及辐射增敏的治疗策略。方法(1)采用转录组测序技术联合q-PCR技术定量检测人正常皮肤组织和人黑色素瘤组织、人黑色素瘤细胞系以及裸鼠移植瘤组织中的PURPL的表达水平;(2)采用荧光原位杂交(FISH)技术和原位杂交技术(ISH),确定PURPL在细胞中与组织中的定位和表达水平;(3)采用细胞克隆形成实验、CCK-8实验、流式细胞术及Transwell迁移和侵袭实验检测细胞增殖、凋亡以及肿瘤细胞的迁移和侵袭能力等生物学功能,并采用Western blot检测生物学功能相关蛋白表达水平;(4)采用免疫荧光检测γ-H2AX、彗星电泳实验、SYTOX染色实验和Western blot检测DNA损伤相关蛋白确定细胞DNA损伤水平;(5)采用RNA pulldown实验收集与PURPL相互作用的蛋白,联合高效液相色谱结合质谱进行鉴定,采用Western blot进行验证;(6)建立裸鼠异体移植瘤模型,并对移植瘤进行精准放疗,验证PURPL的生物学功能以及放疗抗性作用机制假设,记录并绘制肿瘤生长曲线;采用q-PCR定量检测移植瘤中PURPL的表达量;采用Western blot检测DNA损伤相关蛋白表达量及下游凋亡相关蛋白表达量。结果(1)与人正常皮肤组织相比,PURPL呈现为高表达状态(p<0.01);(2)接受辐照处理后的A375细胞中发现PURPL受到电离辐射的显著诱导(p<0.001);(3)与对照组相比,PURPL敲减组细胞增殖能力减弱(p<0.05),细胞凋亡增加(p<0.01),细胞迁移能力(p<0.001)和细胞侵袭能力(p<0.001)减弱;(4)在电离辐射条件系下,PURPL基因沉默后X射线造成的γ-H2AX损伤点增加(p<0.01),彗星电泳拖尾现象增加,DNA损伤增加(p<0.001),尾部DNA含量增加(p<0.001),细胞死亡现象增加(p<0.001);(5)在电离辐射条件下,PURPL与ICAD和BID相互作用以调节细胞凋亡进程;(6)裸鼠移植瘤模型揭示,在非电离辐射条件下,与对照组相比PURPL敲减组的肿瘤生长速度更缓慢(p<0.01);精准放疗后,PURPL敲减组的肿瘤体积更小(p<0.001),DNA损伤相关蛋白和细胞凋亡相关蛋白表达量均有增加。结论(1)PURPL在人黑色素瘤中高表达,发挥癌基因作用;(2)电离辐射诱导PURPL表达,并且可以增加人黑色素瘤的辐射抗性;(3)PURPL与ICAD和BID分别发生直接相互作用,而电离辐射处理可调节PURPL与ICAD和BID相互作用,从而进一步调控细胞凋亡进程;(4)体内实验进一步验证PURPL在人黑色素瘤中的癌基因功能及发挥增强电离辐射抗性的功能与机制。更多还原

【Abstract】 Background With serious environmental pollution,improper sun protection,and the prevalence of sunbathing,the incidence of skin exposure to sunlight has increased,as well as the aging structure of the population.The incidence and mortality of skin melanoma have also increased year by year.Past clinical experience suggests that melanoma is not sensitive to conventional radiotherapy.Objective To explore the biological functions of PURPL in human melanoma and to further clarify the mechanism of PURPL in the radiotherapy resistance of human melanoma,we want to provide theoretical basis and radiation sensitization treatment strategies for the clinical treatment of human melanoma.Methods(1)Transcriptomic sequencing technology combined with q-PCR technology was used to detect the expression levels of PURPL in human normal skin tissues and human melanoma tissues,human melanoma cell lines and xenografts transplanted to nude mice;(2)Fluorescence in situ hybridization(FISH)technology and original hybridization technology were used to determine the location of PURPL in cells and expression levels in different tissues;(3)Colony formation assay,CCK-8 experiment,flow cytometry assay,Transwell and Matrigel invasion assay were used to detect cell proliferation,apoptosis,and tumor cell migration and invasion capabilities and other biological functions,and then Western blot was used to detect the expression levels of biological function-related proteins;(4)Immunofluorescence detection of y-H2AX,comet experiment,SYTOX staining experiment and Western blot to detect DNA damage-related proteins to determine the level of cell DNA damage;(5)RNA pull down experiment and high performance liquid chromatography combined with mass spectrometry was used to detect the proteins interacting with PURPL which were verified by Western blot;(6)Establish a xenograft model in nude mice and perform precise radiotherapy on xenografts,recorded and drew tumor growth curves to verify the biological functions and mechanism of PURPL in mediating radiotherapy resistance;q-PCR quantification was used to detect the expression levels of PURPL in xenografts;Western blot was used to detect DNA damage-related protein expression and downstream apoptosis-related protein expression.Results(1)Compared with normal human skin tissue,PURPL showed relatively high expression levels in melanoma(p<0.01);(2)PURPL was found to be significantly induced by radiation in A375 cells(p<0.001);(3)Compared with control group,the PURPL knock down treatment group decreased cell proliferation(p<0.05),cell migration(p<0.001)and cell invasion(p<0.001)while increased cell apoptotic rate(p<0.01);(4)Under radiation conditions,the y-H2AX foci,comet tailing,tail DNA amount and cell death rate induced by X-rays increased after PURPL gene knockdown(p<0.01);(5)Under radiation conditions,PURPL promotes the process of cell apoptosis by dissociating from ICAD;(6)The xenograft model in mice revealed that under non-irradiation conditions,the tumor growth rate of the PURPL knockdown treatment group was slower than that of control group(p<0.01);after ionizing radiation treatment,the tumor volume of the PURPL knockdown treatment group was smaller(p<0.001)and the expression levels of DNA damage-related protein and apoptosisrelated protein increased.Conclusion(1)PURPL is relatively highly expressed in human melanoma and acts as oncogene;(2)PURPL is induced by ionizing radiation and can enhance the radiation resistance of melanoma cells;(3)PURPL interacts directly with ICAD and BID respectively,and ionizing radiation treatment can adjust the interaction of PURPL with ICAD and BID,thereby further regulating the process of cell apoptosis;(4)The oncogenic and pro-radioresistant function and mechanism of PURPL in human melanoma was verified by in vivo experiments.更多还原