应激诱导蛋白Sestrin2对脂多糖诱发树突状细胞坏死性凋亡的影响  

Effects of stress-induced protein Sestrin2 on necroptosis of dendritic cells induced by lipopolysaccharide

作　　者：吴梦瑶 姚人骐 段昱 王陆 郑丽玉 贺鹏翼 董宁[1] 吴瑶 姚咏明[1] Wu Mengyao;Yao Renqi;Duan Yu;Wang Lu;Zheng Liyu;He Pengyi;Dong Ning;Wu Yao;Yao Yongming(Medical Innovation Research Division of the Chinese PLA General Hospital,Beijing 100853,China;Department of General Surgery,the First Medical Center of the Chinese PLA General Hospital,Beijing 100853,China;Department of Critical Care Medicine,the First Medical Center of the Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]解放军总医院医学创新研究部,北京100853 [2]解放军总医院第一医学中心普通外科,北京100853 [3]解放军总医院第一医学中心重症医学科,北京100853

出　　处：《中华危重病急救医学》2024年第3期237-243,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金重点项目(82241062,82130062)。

摘　　要：目的探讨应激诱导蛋白Sestrin2(SESN2)对脂多糖(LPS)联合泛天冬氨酸特异性半胱氨酸蛋白酶(caspase)抑制剂zVAD诱导小鼠树突状细胞(DC)坏死性凋亡的影响。方法将培养至第3~10代小鼠骨髓来源树突状细胞系DC2.4给予LPS刺激0、6、12、24 h,并按照刺激时间点进行分组,采用蛋白质免疫印迹试验(Western blotting)检测各组细胞SESN2蛋白表达水平。将过表达空载慢病毒(NC)、SESN2基因过表达RNA序列慢病毒(SESN2 LV-RNA)、小干扰空载慢病毒(NS)以及SESN2基因小干扰RNA序列慢病毒(SESN2 siRNA)分别转染至DC2.4细胞中,转染后72 h,于倒置荧光显微镜下观察细胞荧光表达改变。各转染组细胞给予LPS刺激24 h,同时设立空白对照组给予磷酸盐缓冲液(PBS)并培养24 h,采用Western blotting检测SESN2蛋白表达水平;给予LPS+zVAD刺激24 h,同时设立空白对照组给予PBS并培养24 h,采用Western blotting检测混合谱系激酶结构域样蛋白(MLKL)、磷酸化MLKL(p-MLKL)蛋白表达水平,使用激光共聚焦显微镜观察p-MLKL变化及阳性细胞数量,采用流式细胞术和Hoechst染色检测坏死性凋亡细胞比例。结果与LPS刺激0 h组相比,LPS刺激24 h组细胞SESN2蛋白表达明显升高,故将24 h作为后续实验LPS刺激时间点。成功转染慢病毒后培养24 h,SESN2 siRNA+LPS+zVAD组细胞MLKL磷酸化水平明显高于NS+LPS+zVAD组;SESN2 LV-RNA+LPS+zVAD组细胞MLKL磷酸化水平明显低于NC+LPS+zVAD组;且NS+LPS+zVAD组和NC+LPS+zVAD组MLKL磷酸化水平分别高于NS+PBS组、NC+PBS组。激光共聚焦显微镜下显示,各组细胞p-MLKL蛋白荧光表达数量及荧光强度变化趋势与上述结果一致。流式细胞术和Hoechst染色检测结果均显示,SESN2 siRNA+LPS+zVAD组坏死性凋亡细胞比例明显高于NS+LPS+zVAD组〔流式细胞术:(30.800±1.153)%比(20.800±1.114)%,Hoechst染色:(75.267±0.451)%比(46.267±3.371)%,均P<0.05〕,提示敲减SESN2表达可以进一步加剧细胞坏死性凋亡发生Objective To investigate the effect of stress-induced protein Sestrin2(SESN2)on necroptosis of mouse dendritic cell(DC)induced by lipopolysaccharide(LPS)combined with zVAD,a panaspartate-specific cysteine protease(caspase)inhibitor.Methods The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured.The cells were stimulated with LPS for 0 hour,6 hours,12 hours,and 24 hours,and grouped according to the stimulation time points.Western blotting was performed to determine the protein expression of SESN2 in each group.Overexpression empty lentivirus(NC),SESN2 gene overexpression RNA sequence lentivirus(SESN2 LV-RNA),small interfering empty lentivirus(NS),and SESN2 gene small interfering RNA sequence lentivirus(SESN2 siRNA)were transfected into DC2.4 cells.After 72 hours of transfection,cell fluorescence expression was observed under the inverted fluorescence microscope.Cells in each transfection group were stimulated with LPS for 24 hours.The blank control groups were set up and cultured with phosphate buffered saline(PBS)for 24 hours.Western blotting was performed to measure SESN2 protein expression.In the same groups as above,cells were stimulated with LPS+zVAD for 24 hours.The blank control groups were set up and cultured with PBS for 24 hours.Western blotting was used to determine the expression of mixed lineage kinase domain-like protein(MLKL)and phosphorylated-MLKL(p-MLKL).The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy.The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining.Results Compared to the LPS 0 hour group,the expression of SESN2 in the LPS 24 hours group showed a significant increase.Therefore,24 hours was chosen as the subsequent stimulation time point.After successful lentivirus transduction and 24 hours of cultivation,the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group.The MLKL phosphorylation in the

关 键 词：应激诱导蛋白Sestrin2 坏死性凋亡 脓毒症 树突状细胞 免疫反应 

分 类 号：R459.7[医药卫生—急诊医学]