内质网应激PERK-eIF2α-ATF4信号通路在延缓APP/PS1小鼠移植瘤生长中的作用  

Effect of endoplasmic reticulum stress PERK-eIF2α-ATF4 signaling pathway on delaying transplanted tumor growth in APP/PS1 mice

作　　者：董营[1] 郭家女 王思懿 郭丹 王力可 温旭 刘立峰[2] 曲萌[3] 于春艳[1] 刘楠楠[1] 王丹[1] 陈昌捷[4] DONG Ying;GUO Jianyu;WANG Siyi;GUO Dan;WANG Like;WEN Xu;LIU Lifeng;QU Meng;YU Chunyan;LIU Nannan;WANG Dan;CHEN Changjie(Department of Pathology,College of Basic Medical Sciences,Beihua University,Jilin 132013,China;Department of Human Anatomy,College of Basic Medical Sciences,Beihua University,Jilin 132013,China;Department of Pathophysiology,College of Basic Medical Sciences,Beihua University,Jilin 132013,China;Department of Clinical Medicine Laboratory,Affiliated Hospital,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学基础医学院病理教研室,吉林吉林132013 [2]北华大学基础医学院人体解剖学教研室,吉林吉林132013 [3]北华大学基础医学院分子生物教研室,吉林吉林132013 [4]北华大学附属医院检验科,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2022年第2期324-330,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(YDZJ202101ZYTS090,20210101022JC);吉林省教育厅科学技术研究项目(JJKH20210052KJ);吉林省吉林市科技局医疗卫生指导性计划项目(20200404101);吉林省大学生创新创业项目(202010201073,S202110201102);北华大学研究生教改项目(JG2021035)。

摘　　要：目的:探讨内质网应激(ERS)及内质网自噬对移植黑色素瘤模型淀粉样蛋白前体蛋白(APP)/早老素1(PS1)小鼠移植瘤生长的抑制作用,并阐明蛋白激酶R样内质网激酶(PERK)-真核翻译起始因子2α(eIF2α)-活化转录因子4(ATF4)通路在其抑制作用中的可能机制。方法:体外培养B16细胞,通过皮下注射分别植入C57和APP/PS1小鼠背部皮下,建立C57BL/6J和APP/PS1雄性小鼠移植黑色素瘤模型,作为C57移植瘤组(n=7)和APP/PS1移植瘤组(n=7)。观察2组小鼠出瘤时间并计算肿瘤体积,实时荧光定量PCR(RT-qPCR)法检测2组小鼠移植瘤组织中葡萄糖调节蛋白78(GRP78)、PERK和溶酶体组织蛋白酶L(cathepsin L)mRNA表达水平,Western blotting法检测2组小鼠移植瘤组织中GRP78、PERK、磷酸化PERK(p-PERK)、真核翻译起始因子2α(eIF2α)、磷酸化eIF2α(p-eIF2α)、活化转录因子4(ATF4)和内质网自噬标志蛋白FAM134B蛋白表达水平,免疫组织化学法检测移植瘤组织中蛋白质二硫异构酶(PDI)蛋白表达情况,免疫荧光法测定2组小鼠移植瘤组织中腺嘌呤核苷三磷酸(ATP)水平。结果:与C57移植瘤组比较,APP/PS1移植瘤组小鼠出瘤时间晚,肿瘤体积小(P<0.05),移植瘤组织中ERS相关基因GRP78和PERK mRNA表达水平升高(P<0.05),移植瘤组织中GRP78、p-eIF2α/eIF2α和ATF4蛋白表达水平及p-PERK/PERK比值均升高(P<0.05)。C57移植瘤组肿瘤细胞胞质内多见黑色素颗粒,为移植瘤组织的形态特征,可见少量棕黄色颗粒,为PDI阳性颗粒;APP/PS1移植瘤组肿瘤细胞胞质内可见少量黑色素颗粒和广泛表达的棕黄色颗粒。与C57移植瘤组比较,APP/PS1移植瘤组小鼠移植瘤组织中内质网自噬标志蛋白FAM 134B蛋白表达水平升高(P<0.05),cathepsin L mRNA表达水平升高(P<0.05),ATP水平降低(P<0.05)。结论:APP/PS1移植瘤模型小鼠肿瘤生长缓慢,其机制可能与ERS的PERK-eIF2α-ATF4信号通路被激活有关。Objective:To investigate the inhibitory effects of endoplasmic reticulum stress(ERS)and endoplasmic reticulum autophagy on the growth of transplanted melanoma model in amyloid precursor protein(APP)/presenilin 1(PS1)mice,and to clarify the possible mechanism of its inhibitory effect of protein kinase R-like endoplasmic reticulum(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(RATF4)pathway.Methods:The C57BL/6J(C57)and APP/PS1 mice were transplanted with melanoma B16 cells to establish the C57 transplanted tumor and APP/PS1transplanted tumor models of male mice),and used as C57 transplanted tumor group(n=7)and APP/PS1transplanted tumor group(n=7).The tumor appearance time was observed and the tumor volume was calculated of the mice in two groups.The expression levels of glucose regulatory protein 78(GRP78),PERK and lysosomal cathepsin L(cathepsin L)mRNA in the transplanted tumor tissue of the mice in two groups were detected by real-time fluorescence quantitative PCR(RT-PCR).The expression levels of GRP78,PERK,phos-PERK,eukaryotic translation initiation factor 2α(eIF2α),p-eIF2α,ATF4,and FAM134B in the transplanted tumor tissue of the mice in two groups were detected by Western blotting method.The expression levels of protein disulfide isomerase(PDI)protein in two groups were detected by immunohistochemistry and the adenosine triphoshate(ATP)levels in the transplanted tumor tissue of the mice were determined by fluorescence assay.Results:Compared with C57 transplanted tumor group,the tumor appearance time of the mice in APP/PS1 transplanted tumor group was late,and the tumor volume was decreased(P<0.05);the expression levels of GRP78 and PERK mRNA in the transplanted tumor tissue were increased(P<0.05),and the expression levels of GRP78,p-PERK/PERK,p-eIF2α/eIF2αand ATF4 proteins were increased(P<0.05).In C57 transplanted tumor group,melanoma granules were found in the cytoplasm of the tumor cells,which was the morphological characteristic of transplanted tumor tissu

关 键 词：蛋白激酶R样内质网激酶 内质网应激 内质网自噬 淀粉蛋白前体蛋白 早老素1 移植黑色素瘤 

分 类 号：R739.5[医药卫生—肿瘤]