干扰素刺激因子对脂多糖诱导的小鼠树突状细胞坏死性凋亡的影响  

The influence of stimulator of interferon genes on lipopolysaccharide-induced necroptosis of mouse dendritic cells

作　　者：李金儒 段昱 凌花 李琼 姚咏明[3] 戴新贵 Li Jinru;Duan Yu;Ling Hua;Li Qiong;Yao Yongming;Dai Xingui(The First School of Clinical Medicine,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学第一临床医学院,广东广州510515 [2]南方医科大学附属郴州医院(郴州市第一人民医院)重症医学科,湖南郴州423000 [3]解放军总医院医学创新研究部转化医学研究中心第四医学中心,北京100853

出　　处：《中国急救医学》2024年第7期611-617,共7页Chinese Journal of Critical Care Medicine

基　　金：国家自然科学基金项目重点专项(82241062);湖南省卫健委科研项目(B202317017622);湘南学院科研课题(2020XJ89)。

摘　　要：目的 探讨干扰素刺激因子(STING)对脂多糖(LPS)诱导的小鼠树突状细胞(DC)坏死性凋亡的影响。方法 LPS 1μg/mL刺激第3~10代对数生长期小鼠DC2.4细胞系,将DC分为对照组、LPS 6 h组、LPS 12 h组、LPS 24 h组和LPS 72 h组,采用蛋白质印迹法(Western blot)检测磷酸化干扰素刺激因子(P-STING)、STING、磷酸化混合系激酶区域样蛋白(P-MLKL)、MLKL蛋白的表达。将转染含STING基因小干扰RNA序列慢病毒的DC分为敲减STING+磷酸盐缓冲液(PBS)组、敲减STING+LPS组,将转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予相应刺激并培养12 h,通过流式细胞术检测细胞凋亡率,行核酸染料(SYTOX Green)染色观测细胞凋亡情况并计算凋亡率,Western blot检测P-STING、STING、P-MLKL、MLKL蛋白表达。结果 与对照组比较,LPS 12 h组STING蛋白表达显著升高(P<0.05),LPS 12 h组、LPS 24 h组P-MLKL/MLKL比值均显著升高(P<0.05),将12 h作为后续刺激时长。培养12 h,与PBS组比较,LPS组细胞的凋亡率显著上升,且LPS组细胞器普遍肿胀,细胞质中出现大量空泡,细胞膜完整性被破坏,细胞发生崩解。空载体组与敲减STING组细胞转染慢病毒72 h后,Western blot检测提示,sh1-STING敲减组STING蛋白较空载体组、sh2-STING敲减组和sh3-STING敲减组表达显著降低(P<0.05)。培养12 h,流式细胞术检测显示,空载体+PBS组、空载体+LPS组、sh1-STING敲减+PBS组、sh1-STING敲减+LPS组细胞凋亡率分别为(36.34±24.42)%、(52.57±35.77)%、(37.81±28.9)%、(46.45±32.44)%。培养12 h, SYTOX Green染色检测显示,空载体+PBS组、空载体+LPS组、sh1-STING敲减+PBS组、sh1-STING敲减+LPS组细胞凋亡率分别为(13.4±6.3)%、(67.6±24.0)%、(2.0±1.1)%和(7.1±4.2)%。培养12 h,与空载体+LPS组比较,sh1-STING敲减+LPS组细胞凋亡率以及细胞中P-MLKL/MLKL比值均显著降低(P<0.05);与空载体+PBS组比较,空载体+LPS组P-MLKL/MLKL比值明显升高(P=0.011);但与敲减STING+PBSObjective To investigate the influence of stimulator of interferon genes(STING)on lipopolysaccharide(LPS)-induced necroptosis of dendritic cells(DCs).Methods The DC 2.4 cell line of the 3rd to 10th passage in the logarithmic growth stage stimulated by 1μg/mL LPS were divided into Control group,LPS 6 h group,LPS 12 h group,LPS 24 h group,LPS 72 h group.Western blot was used to determine the expression of phosphorylated(P)-STING,STING,phosphorylated-mixed lineage kinase domain-like protein(P-MLKL),MLKL in DCs.DCs transfected with lentivirus containing siRNA sequence of STING gene were divided into STING-knockdown+phosphate buffer solution(PBS)group and STING-knockdown+LPS group,and DCs transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group.After LPS stimulation and cultivation for 12 h,the apoptotic rate of cells was determined by flow cytometry,the situation of apoptosis was observed by SYTOX Green staining and the apoptotic rate was calculated,the protein expression of P-STING,STING,P-MLKL and MLKL was detected by using Western blot.Results Compared with those in control group,the protein expression of STING of cells in LPS 12 h group was obviously increased(P<0.05),and the ratios of P-MLKL/MLKL of cells in LPS 12 h group and LPS 24 h group were significantly increased(P<0.05),therefore 12 hours were used as the subsequent LPS stimulation duration.After 12 hours of culture,compared with PBS group,LPS group showed increased necroptosis rate.Furthermore,cells in LPS group exhibited general organelle swelling,the abundant vacuoles in the cytoplasm,the disruption of cell membrane integrity,and cellular disintegration.After 72 hours of transfection,compared with empty vector group,sh2-STING-knockdown group and sh3-STING-knockdown group,the expression of STING determined by Western blot in DCs was significantly decreased in sh1-STING-knockdown group(P<0.05).After 12 hours of culture,flow cytometry assay revealed that the apoptotic rate of cells in empty vector+PBS group,e

关 键 词：脓毒症 树突细胞 坏死性凋亡 干扰素刺激因子 免疫功能障碍 

分 类 号：R459.7[医药卫生—急诊医学]