紫外线和人乳头状瘤病毒16 E6协同诱导、促进裸鼠皮肤鳞状细胞癌的机制研究  

Mechanisms underlying synergistic induction and promotion of cutaneous squamous cell carcinoma in nude mice by ultraviolet light and human papillomavirus 16 E6

作　　者：唐洪波 马庆宇 桑莹冰 毛丽旦 梁俊琴[1] 康晓静[1] Tang Hongbo;Ma Qingyu;Sang Yingbing;Mao Lidan;Liang Junqin;Kang Xiaojing(Department of Dermatology and Venereology,People′s Hospital of Xinjiang Uygur Autonomous Region,Xinjiang Clinical Research Center for Dermatologic Diseases,Xinjiang Key Laboratory of Dermatology Research(XJYS1707),Urumqi 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院皮肤性病科,新疆皮肤病临床医学研究中心,新疆皮肤病研究重点实验室(XJYS1707),乌鲁木齐830002

出　　处：《中华皮肤科杂志》2022年第11期982-989,共8页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81760494)。

摘　　要：目的:构建裸鼠皮肤鳞状细胞癌(CSCC)移植瘤模型,探讨紫外线(UV)损伤及人乳头瘤病毒(HPV)感染诱导、促进CSCC的协同作用机制。方法:将人CSCC细胞A431分成3组,即用HPV16 E6腺病毒转染的HPV16 E6过表达组,空白腺病毒转染的空白载体组(简称空载组),未进行腺病毒转染的空白对照组。使用无血清DMEM培养基将空载组及HPV16 E6过表达组(LV-OE-HPV16 E6组)A431细胞制成单细胞悬液,分别接种于SKH-1裸鼠左侧臀部皮下作为空载组(n=16)和LV-OE-HPV16 E6组(n=16)。每3天观察并记录小鼠肿瘤生长情况,当瘤体达到150 mm 3时,视为建模成功。建模成功后,每组取8只小鼠进行UV照射,分为4组,即空载组、空载+UV组、LV-OE-HPV16 E6组、LV-OE-HPV16 E6+UV组,UV照射剂量为1440 mJ/(cm 2·d),每次12 min,持续4周后处死裸鼠,测量瘤重及体积,绘制肿瘤生长曲线,免疫组化、Western印迹和qRT-PCR检测验证Wnt1、β联蛋白mRNA及蛋白在裸鼠CSCC中的表达。数据若符合正态分布,多组间比较采用方差分析,组间两两比较采用LSD-t检验;数据若不符合正态分布,采用秩和检验对数据进行统计分析。结果:空载+UV组瘤重为(2.90±0.36)g,LV-OE-HPV16 E6组(3.19±0.32)g,LV-OE-HPV16 E6+UV组(4.41±0.18)g,与空载组(2.20±0.24)g比较,差异均有统计学意义(t值分别为4.39、6.77、20.11,均P<0.001);空载+UV组瘤体积为(1033.12±400.15)mm 3,LV-OE-HPV16 E6组(1119.21±447.57)mm 3,LV-OE-HPV16 E6+UV组(1464.29±409.98)mm 3,与空载组(688.94±319.31)mm 3比较差异均有统计学意义(t值分别为1.90、2.21、4.22,均P<0.001)。免疫组化显示,4组间Wnt1、β联蛋白表达水平差异无统计学意义(F值分别为0.76、0.71,均P>0.05);Western印迹显示,4组间Wnt1、β联蛋白水平差异有统计学意义(F值分别为16.74、49.90,均P<0.05),且LV-OE-HPV16 E6+UV组Wnt1、β联蛋白水平高于空载组、空载+UV组和LV-OE-HPV16 E6组(均P<0.05)。mRNA水平分析显示,4组间组织中Wnt1、β联蛋白mObjective To establish a xenograft model of cutaneous squamous cell carcinoma(CSCC)in nude mice,and to explore mechanisms underlying synergistic induction and promotion of CSCC in nude mice by ultraviolet radiation and human papillomavirus(HPV)infection.Methods The human CSCC A431 cells were divided into 3 groups,namely HPV16 E6 overexpression group(LV-OE-HPV16 E6 group)transfected with adenovirus-mediated HPV16 E6 gene,empty vector group transfected with empty adenovirus vectors,and blank control group remaining untransfected.Using serum-free Dulbecco′s modified Eagle′s medium(DMEM),A431 cells in the empty vector group and LV-OE-HPV16 E6 group were prepared into single-cell suspensions,which were subcutaneously inoculated into the left buttocks of SKH-1 nude mice separately,namely empty vector group(n=16)and LV-OE-HPV16 E6 group(n=16).Tumor growth was observed and recorded for the mice every 3 days.When the tumor size reached 150 mm3,the modeling was considered successful.After successful modeling,8 mice in each group were irradiated with ultraviolet light at a dose of 1440 mJ·cm-2·d-1 for 12 minutes each time,the other 8 mice in each group received no ultraviolet radiation,that is to say,all the 32 mice were divided into 4 groups:empty vector group,empty vector+UV group,LV-OE-HPV16 E6 group,and LV-OE-HPV16 E6+UV group.After 4-week radiation,these nude mice were sacrificed,tumor weight and volume were measured,a tumor growth curve was drawn,immunohistochemistry study,Western blot analysis and real-time fluorescence-based quantitative PCR(qRT-PCR)were conducted to determine the protein and mRNA expression of Wnt1 andβ-catenin in CSCC tissues collected from nude mice,respectively.For normally distributed measurement data,analysis of variance was used for intergroup comparisons,and least significant difference-t test for multiple comparisons;for non-normally distributed measurement data,rank sum test was used for intergroup comparisons.Results Compared with the empty vector group(2.20±0.24 g),the tumor weig

关 键 词：肿瘤 鳞状细胞 疾病模型 动物 紫外线 人乳头瘤病毒16 HPV16 E6 协同作用 

分 类 号：R739.5[医药卫生—肿瘤]