序列相似性家族134成员B介导的内质网自噬对内毒素/脂多糖诱导的小鼠树突状细胞凋亡的影响  

Influence of family with sequence similarity 134,member B-mediated reticulophagy on lipopolysaccharide-induced apoptosis of mouse dendritic cells

作　　者：段昱 姚人骐 郑丽玉 董宁[2] 吴瑶 姚咏明[2] 戴新贵 Duan Yu;Yao Renqi;Zheng Liyu;Dong Ning;Wu Yao;Yao Yongming;Dai Xingui(Department of Critical Care Medicine,Affiliated Chenzhou Hospital,Southern Medical University(The First People's Hospital of Chenzhou),Chenzhou 423000,China;Translational Medicine Research Center,Medical Innovation Research Division and the Fourth Medical Center of PLA General Hospital,Beijing 100853,China)

机构地区：[1]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,郴州423000 [2]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：《中华烧伤与创面修复杂志》2023年第9期857-866,共10页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金重点项目(82130062,82241062)。

摘　　要：目的探讨序列相似性家族134成员B(FAM134B)介导的内质网自噬对内毒素/脂多糖(LPS)诱导的小鼠树突状细胞(DC)凋亡的影响,为改善创面感染等引起的脓毒症免疫抑制提供依据。方法采用实验研究方法。取第3~10代对数生长期的小鼠DC系DC2.4进行实验。将DC分为LPS刺激0 h(不刺激)组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激24 h组和LPS刺激72 h组,采用1μg/mL LPS(浓度下同)培养相应时间后,采用蛋白质印迹法检测FAM134B、微管相关蛋白1轻链3B(LC3B)以及转运蛋白SEC61B蛋白表达,并计算LC3B-Ⅱ/LC3B-Ⅰ比值(样本数为3)。将DC分为进行相应处理的磷酸盐缓冲液(PBS)组与LPS组,培养24 h,采用免疫荧光法检测FAM134B表达情况及其分别与溶酶体探针、LC3B共定位情况,通过透射电子显微镜观察细胞内自噬溶酶体数量。将DC分为转染含FAM134B基因小干扰RNA序列的慢病毒的敲减FAM134B组与转染空载慢病毒的空载体组,转染后72 h,于倒置荧光相差显微镜下观察细胞荧光表达情况,另将仅常规培养的DC设为空白对照组并于相应时间点行相同观察。将DC分为单纯PBS组、单纯LPS组,将成功转染含FAM134B基因小干扰RNA序列的慢病毒的DC分为敲减FAM134B+PBS组、敲减FAM134B+LPS组,将成功转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予相应刺激并培养24 h,采用蛋白质印迹法检测FAM134B蛋白表达(样本数为3),通过流式细胞术检测细胞凋亡率(样本数为3),行Hoechst染色观测细胞凋亡情况并计算凋亡率(样本数为5),采用蛋白质印迹法检测剪切型胱天蛋白酶3(caspase-3)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达并计算Bax/Bcl-2比值(样本数为5)。对数据行单因素方差分析、LSD检验、析因设计方差分析。结果与LPS刺激0 h组相比,LPS刺激12 h组和LPS刺激24 h组细胞中FAM134B蛋白表达均明显升高(P<0.05),LPS刺激6 h组、LPS刺激12 h组、LPS刺激24Objective To investigate the influence of family with sequence similarity 134,member B(FAM134B)-mediated reticulophagy on lipopolysaccharide(LPS)-induced apoptosis of mouse dendritic cells(DCs),so as to provide a basis for improving the immune suppression of sepsis caused by wound infection and other factors.Methods The experimental research methods were used.The DC line DC2.4 of the 3^(rd) to 10^(th) passage in the logarithmic growth stage was collected for experiments.DCs were divided into LPS stimulation 0 h(no stimulation)group,LPS stimulation 6 h group,LPS stimulation 12 h group,LPS stimulation 24 h group,and LPS stimulation 72 h group,which were cultured with 1μg/mL LPS(the same concentration below)for the corresponding time.The protein expressions of FAM134B,microtubule-associated protein 1 light chain 3B(LC3B),and transporter protein SEC61B were determined by Western blotting,and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated(n=3).DCs were divided into phosphate buffer solution(PBS)group and LPS group for corresponding treatment.After 24 hours of culture,the expression of FAM134B and its co-localization with lysosomal probes and LC3B were detected using immunofluorescence method,while the number of autolysosomes in cells were observed through transmission electron microscope.DCs were divided into the FAM134B-knockdown group that were transfected with lentivirus containing small interfering RNA(siRNA)sequence of FAM134B gene and the empty vector group with empty lentivirus transfected.At post transfection hour 72,the fluorescence expression of cells was observed under the inverted fluorescence phase contrast microscope,meanwhile,the normally cultured DCs were set as blank control group,and the same observation was performed at the corresponding time point.DCs were divided into PBS alone group and LPS alone group,DCs successfully transfected with lentivirus containing siRNA sequence of FAM134B gene were divided into FAM134B-knockdown+PBS group and FAM134B-knockdown+LPS group,and DCs successfully transfected

关 键 词：脓毒症 树突细胞 细胞凋亡 序列相似性家族134成员B 内质网自噬 免疫功能障碍 

分 类 号：R459.7[医药卫生—急诊医学]