【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。更多还原

【Abstract】 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 tissue,and a small amount of brown granules were found,which were PDI positive granules. In APP/PS1transplanted tumor group,a small amount of melanoma granules and widely expressed brown granules were found in the cytoplasm of tumor cells. Compared with C57 transplanted tumor group,the expression level of FAM 134B protein in the transplanted tumor tissue of the mice in APP/PS1 transplanted tumor group was increased(P<0. 05),the expression level of cathepsin L mRNA was increased(P<0. 05),and the ATP level was decreased(P<0. 05). Conclusion:The growth of tumor in the APP/PS1 transplanted tumor model mice is slow,and its mechanism may be related to the activation of endoplasmic reticulum stress PERK-eIF2α-ATF4 signaling pathway and regulated endoplasmic reticulum autophagy.更多还原