沉默GRP78表达增强HOS细胞对MPPα-PDT敏感性的研究  

Silencing GRP78 expression enhances sensitivity of HOS cells to MPPα-PDT therapy

作　　者：左强 欧云生[1] 余浩洋 钟申熹 张木子 ZUO Qiang;OU Yun-Sheng;YU Hao-Yang;ZHONG Shen-Xi;ZHANG Mu-Zi(Department of Orthopedic Surgery,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016

出　　处：《中国免疫学杂志》2021年第14期1665-1671,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(81572634)。

摘　　要：目的:观察葡萄糖调节蛋白78(GRP78)在焦脱镁叶绿酸-α甲酯(MPPα)介导的光动力疗法(PDT)(MPPα-PDT)处理骨肉瘤HOS细胞中的作用,并探讨沉默GRP78表达增强HOS细胞对MPPα-PDT敏感性的可能机制。方法:MPPα-PDT作用于HOS细胞24 h后,Hoechst染色观察胞核形态学变化。采用Western blot检测MPPα-PDT 3 h、6 h、12 h和24 h处理HOS细胞后GRP78蛋白的表达水平;运用脂质体法将特异性针对人siRNA-GRP78转染HOS细胞后,分为Control组、siRNAGRP78组、siRNA-GRP78+MPPα-PDT组及MPPα-PDT组,细胞免疫荧光法检测GRP78表达,CCK-8检测HOS细胞增殖活性,Western blot检测细胞增殖相关蛋白、凋亡相关蛋白及Wnt通路相关蛋白的表达;流式细胞术检测各组细胞的凋亡率。结果:MPPα-PDT可诱导骨肉瘤HOS细胞凋亡及GRP78表达增加;有效转染siRNA-GRP78后,HOS细胞中GRP78的mRNA及蛋白表达均下降,GRP78平均荧光强度降低(P<0.05),细胞增殖蛋白PCNA、Ki67表达水平下降(P<0.05),凋亡相关蛋白CleavedPARP、Cleaved-Caspase 3的表达水平上调(P<0.05),Wnt通路相关蛋白表达下降。结论:MPPα-PDT可诱导骨肉瘤HOS细胞凋亡及GRP78表达增加;siRNA-GRP78有效沉默GRP78表达后,HOS细胞增殖活性降低,凋亡水平增加,并增加HOS细胞对MPPα-PDT的敏感性,其机制可能与抑制Wnt通路激活相关。Objective:To observe the role of glucose regulated protein 78(GRP78)in the treatment of pyropheophorbide-αmethyl ester-mediated photodynamic therapy(MPPα-PDT)on human osteosarcoma HOS cells,and to explore the possible mechanism of silencing GRP78 expression to enhance the sensitivity of HOS osteosarcoma cells to MPPα-PDT.Methods:The HOS apoptotic morphology after MPPα-PDT 24 h was observed by Hoechst staining.The expression level of GRP78 protein in HOS cells treated with MPPα-PDT for 3 h,6 h,12 h and 24 h was detected by Western blot.The siRNA targeting GRP78 gene was transfected into HOS cells by lipofection,and the groups were divided into control group,siRNA-GRP78 group,siRNA-GRP78+MPPα-PDT group and MPPα-PDT group,the expression of GRP78 was detected by immunofluorescence,the proliferation of HOS cells was detected by CCK-8 assay and the proliferation protein PCNA and Ki67 were detected by Western blot.The apoptosis rate was analyzed by flow cytometry.The expressions of Cleaved-PARP,Cleaved-Caspase 3 and Wnt signaling pathway related proteins were detected by Western blot.The expression of GRP78 was also investigated by immunofluorescence.Results:MPPα-PDT could induce osteosarcoma HOS cell apoptosis and increase GRP78 expression.After successful transfection with siRNA-GRP78,the mRNA and protein expression of GRP78 in HOS cells were decreased,the mean fluorescence intensity of GRP78 decreased(P<0.05),HOS cell proliferation was decreased(P<0.05),the cell proliferation related proteins were decreased(P<0.05),the apoptosis-related proteins were increased(P<0.05),Wnt signaling pathway related proteins were decreased(P<0.05).Conlusion:MPPα-PDT could induce osteosarcoma HOS cell apoptosis and increase GRP78 expression,silencing GRP78 reduces the proliferation activity of HOS osteosarcoma cells,up-regulates the level of apoptosis,and increases the sensitivity of osteosarcoma cells to MPPα-PDT.The mechanism may be related to inhibition of Wnt pathway activation.

关 键 词：葡萄糖调节蛋白78 骨肉瘤 光动力疗法 焦脱镁叶绿酸-α甲酯 WNT信号通路 

分 类 号：R738.1[医药卫生—肿瘤]