基于低糖低血清营养胁迫构建内质网应激和自噬模型及其评价  

Construction and evaluation of endoplasmic reticulum stress and autophagy models based on low-glucose and low-serum nutrient stress

作　　者：化维 朱嫚嫚 刘加涛[1] Hua Wei;Zhu Manman;Liu Jiatao(Dept of Pharmacy,The First Affiliated Hospital of Anhui Medical University, Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院药剂科,合肥230022

出　　处：《安徽医科大学学报》2021年第11期1779-1784,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82072687);安徽省自然科学基金(编号:2008085MH257);高校优秀青年人才支持计划(编号:gxyq2020008)。

摘　　要：目的通过营养胁迫构建内质网应激和自噬模型并评价其对肿瘤增殖和凋亡的影响。方法以含1%血清的低糖DMEM培养基分别处理HepG2细胞、Huh7细胞不同时间,Western blot检测内质网应激标志蛋白葡萄糖调节蛋白78(GRP78)、活化转录因子-6(ATF6)、必需肌醇1α(IRE1α)、蛋白激酶RNA样内质网激蛋白(PERK)和自噬蛋白轻链3(LC3)、核孔蛋白62(P62)、自噬相关蛋白1(Beclin-1)的表达。免疫荧光双染观察自噬蛋白LC3和GRP78的共表达情况。分别采用流式细胞术和CCK-8检测营养胁迫对细胞凋亡和增殖的影响。结果Western blot结果显示营养胁迫可时间依赖性的增加肝癌细胞内质网应激和自噬相关蛋白的表达,且在36 h达到高峰。激光共聚焦检测结果进一步证明营养胁迫36 h后肝癌细胞GRP78和LC3荧光强度显著增加。流式细胞术结果显示,低糖低血清营养胁迫模型不会增加肝癌细胞凋亡,但CCK-8实验提示营养胁迫可抑制肝癌细胞的增殖。结论1%血清低糖DMEM不仅可以诱导内质网应激和自噬且不增加肿瘤细胞凋亡,更符合微环境肿瘤细胞生存状态,为研究内质网应激和自噬提供一种新方法。Objective To construct the models of endoplasmic reticulum stress and autophagy through nutrient stress,and evaluate its effects on tumor proliferation and apoptosis.Methods HepG2 cells and Huh7 cells were treated with low-glucose DMEM containing 1%serum for different time.The endoplasmic reticulum stress marker proteins glucose regulatory protein 78(GRP78),activated transcription factor-6(ATF6),inositol requires 1α(IRE1α),include protein kinase RNA-like ER kinase(PERK)and autophagy-related proteins light chain 3(LC3),nucleoporin 62(P62),autophagy-related protein 1(Beclin-1)were detected by Western blot.Immunofluorescence was used to observe the co-localization of LC3 and GRP78.Flow cytometry was used to detect cell apoptosis rate.CCK-8 was used to detect cell proliferation.Results Western blot showed that nutrient stress increased the expressions levels of GRP78,ATF6,IRE1α,PERK,LC3,P62 and Beclin-1 in HepG2 cells and Huh7 cells in a time-dependent manner,and reached a peak at 36 h.Laser confocal microscopy further showed that the fluorescence intensity of GRP78 and LC3 significantly increased at 36 h.The flow cytometry showed that the low-glucose DMEM containing 1%serum did not increase the apoptosis of liver cancer cells.However,CCK-8 experiments found that nutrient stress could inhibit the proliferation of liver cancer cells.Conclusion Low-glucose DMEM containing 1%serum can not only induce endoplasmic reticulum stress and autophagy,but also not increase tumor cell apoptosis.The models are consistent with the real survival status of tumor cells,and provide a new and better method for studying endoplasmic reticulum stress and autophagy.

关 键 词：营养胁迫 内质网应激 自噬 增殖 凋亡 

分 类 号：R735.7[医药卫生—肿瘤] Q2-33[医药卫生—临床医学]