Influence of family with sequence similarity 134, member B-mediated reticulophagy on lipopolysaccharide-induced apoptosis of mouse dendritic cells
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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1 μg/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24 h组和LPS刺激72 h组细胞中SEC61B蛋白表达均明显降低（ P<0.05），LPS刺激24 h组和LPS刺激72 h组细胞中LC3B-Ⅱ/LC3B-Ⅰ比值均明显升高（ P<0.05），其中LPS刺激24 h组细胞中3个蛋白的变化最显著，将24 h作为后续LPS刺激时长。培养24 h，与PBS组相比，LPS组细胞中FAM134B的表达及其与LC3B及溶酶体探针的共定位均显著增强，自噬溶酶体数量明显增多。空载体组与敲减FAM134B组细胞转染后72 h均表现出高强度荧光，而空白对照组细胞在相应时间点无荧光表现。培养24 h，敲减FAM134B+PBS组细胞中FAM134B蛋白表达较单纯PBS组和空载体+PBS组明显降低（ P值均<0.05），敲减FAM134B+LPS组细胞中FAM134B蛋白表达较单纯LPS组和空载体+LPS组显著降低（ P值均<0.05），单纯LPS组细胞中FAM134B蛋白表达较单纯PBS组明显升高（ P<0.05），空载体+LPS组细胞中FAM134B蛋白表达较空载体+PBS组明显升高（ P<0.05）。培养24 h，流式细胞术检测显示，单纯PBS组、单纯LPS组、空载体+PBS组、空载体+LPS组、敲减FAM134B+PBS组与敲减FAM134B+LPS组细胞凋亡率分别为（13.3±0.8）%、（32.6±4.3）%、（17.0±1.5）%、（51.7±3.3）%、（52.4±3.1）%与（62.3±2.6）%。培养24 h，与单纯LPS组和空载体+LPS组相比，敲减FAM134B+LPS组经流式细胞术与Hoechst染色检测的细胞凋亡率以及细胞中剪切型caspase-3蛋白表达、Bax/Bcl-2比值均明显升高（ P<0.05）；与对应的PBS处理组即单纯PBS组、空载体+PBS组、敲减FAM134B+PBS组相比，单纯LPS组、空载体+LPS组、敲减FAM134B+LPS组经流式细胞术与Hoechst染色检测的细胞凋亡率以及细胞中剪切型caspase-3蛋白表达、Bax/Bcl-2比值均明显升高（ P<0.05）。 结论 小鼠DC中FAM134B介导的内质网自噬在LPS刺激下活化增强并于24 h达活化高峰，且活化的内质网自噬对细胞凋亡具有显著抑制效应。 Abstract:Objective 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 1 μg/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 FAM134Bgene 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 with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. These cells were stimulated correspondingly and cultured for 24 hours. The protein expression of FAM134B was detected using Western blotting ( n=3); the apoptotic rate of cells was determined by flow cytometry ( n=3); the situation of apoptosis was observed by Hoechst staining, and the apoptotic rate was calculated ( n=5); the protein expressions of cleaved cysteine aspartic acid specific protease-3 (caspase-3), B cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were detected using Western blotting, and the ratio of Bax/Bcl-2 was calculated ( n=5). Data were statistically analyzed with one-way analysis of variance (ANOVA), least significant difference test, and ANOVA for factorial design. Results Compared with those in LPS stimulation 0 h group, the protein expressions of FAM134B of cells in LPS stimulation 12 h group and LPS stimulation 24 h group were significantly increased ( P<0.05), the protein expressions of SEC61B of cells in LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group were significantly decreased ( P<0.05), and the ratios of LC3B-Ⅱ/LC3B-Ⅰ of cells in LPS stimulation 24 h group and LPS stimulation 72 h group were obviously increased ( P<0.05). As the most significant changes of three proteins were seen in the cells of LPS stimulation 24 h group, 24 h was used as the duration of subsequent LPS stimulation. After 24 hours of culture, the expression of FAM134B and its co-localization with LC3B and lysosomal probes in the cells of LPS group were all significantly enhanced, with a significant increase in the number of autolysosomes in comparison with those in PBS group. Both the empty vector group and the FAM134B-knockdown group showed high intensity fluorescence in the cells at post transfection hour 72, but the blank control group showed no fluorescence in the cells at the corresponding time point. After 24 hours of culture, the protein expression of FAM134B of cells in FAM134B-knockdown+PBS group was significantly lower than the expressions in PBS alone group and empty vector+PBS group (with P values all <0.05), the protein expression of FAM134B of cells in FAM134B-knockdown+LPS group was significantly lower than the expressions in LPS alone group and empty vector+LPS group (with P values all <0.05), the protein expression of FAM134B of cells in LPS alone group was significantly higher than that in PBS alone group ( P<0.05), while the protein expression of FAM134B of cells in empty vector+LPS group was significantly higher than that in empty vector+PBS group ( P<0.05). After 24 hours of culture, flow cytometry assay revealed that the apoptotic rate of cells in PBS alone group, LPS alone group, empty vector+PBS group, empty vector+LPS group, FAM134B-knockdown+PBS group, and FAM134B-knockdown+LPS group were (13.3±0.8)%, (32.6±4.3)%, (17.0±1.5)%, (51.7±3.3)%, (52.4±3.1)%, and (62.3±2.6)%, respectively. After 24 hours of culture, compared with those in LPS alone group and empty vector+LPS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in FAM134B-knockdown+LPS group ( P<0.05); compared with those in the corresponding PBS treatment group, namely, PBS alone group, empty vector+PBS group, and FAM134B-knockdown+PBS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in LPS alone group, empty vector+LPS group, and FAM134B-knockdown+LPS group ( P<0.05). Conclusions The activation of reticulophagy mediated by FAM134B in mouse DCs is enhanced and peaked in 24 hours under LPS stimulation, and the activated reticulophagy has a significant inhibitory effect on cell apoptosis. -
参考文献
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1 蛋白质印迹法检测的5组小鼠树突状细胞（DC）系DC2.4中内质网自噬相关蛋白的蛋白表达
注：FAM134B为序列相似性家族134成员B，LC3B为微管相关蛋白1轻链3B；条带上方1、2、3、4、5分别指示内毒素/脂多糖（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
2 2组小鼠树突状细胞（DC）系DC2.4培养24 h FAM134B介导的内质网自噬情况 Alexa Fluor 647-Alexa Fluor 488×600。2A、2B.分别为磷酸盐缓冲液（PBS）组、内毒素/脂多糖（LPS）组FAM134B与LC3B共定位情况，图2B中FAM134B表达及其与LC3B的共定位较图2A明显增强；2C、2D.分别为PBS组、LPS组FAM134B与溶酶体探针共定位情况，图2D中FAM13B表达及其与溶酶体探针的共定位较图2C明显增强
注：各图中右上小图为细胞核染色（蓝色）图，右中小图为序列相似性家族134成员B（FAM134B）染色（绿色）图，右下小图为微管相关蛋白1轻链3B（LC3B）或溶酶体探针染色（均为红色）图，各图中左侧大图为右侧3张小图的合成图；FAM134B与LC3B共定位代表其与自噬小体结合情况，FAM134B与溶酶体探针共定位代表其与溶酶体结合情况；图2A、2B为加入FAM134B与LC3B双抗后的染色，图2C、2D为加入FAM134B抗体与溶酶体探针后的染色
3 2组小鼠树突状细胞（DC）系DC2.4培养24 h自噬溶酶体数量 透射电子显微镜×1 000。3A、3B.分别为磷酸盐缓冲液组、内毒素/脂多糖组，与图3A相比，图3B细胞中小空泡状的自噬溶酶体数量明显增多
4 2个转染组小鼠树突状细胞（DC）系DC2.4转染后72 h与空白对照组小鼠DC系DC2.4相应时间点荧光表达 绿色荧光蛋白×50。4A、4B、4C.分别为空白对照组、转染空载慢病毒的空载体组与转染含序列相似性家族134成员B（ FAM134B）基因小干扰RNA序列的慢病毒的敲减FAM134B组，图4A无荧光，图4B、4C表现出明显的绿色荧光，且图4B与图4C间荧光强度无明显差别
5 蛋白质印迹法检测的6组小鼠树突状细胞（DC）系DC2.4培养24 h FAM134B蛋白表达
注：FAM134B为序列相似性家族134成员B；条带上方1、2、3、4、5、6分别指示单纯磷酸盐缓冲液（PBS）组、单纯内毒素/脂多糖（LPS）组、空载体+PBS组、空载体+LPS组、敲减FAM134B+PBS组、敲减FAM134B+LPS组；2个空载体组转染空载慢病毒，2个敲减组转染含FAM134B基因小干扰RNA序列的慢病毒
6 6组小鼠树突状细胞（DC）系DC2.4培养24 h凋亡情况 Hoechst×200。6A、6B、6C、6D、6E、6F.分别为单纯磷酸盐缓冲液（PBS）组、单纯内毒素/脂多糖（LPS）组、空载体+PBS组、空载体+LPS组、敲减FAM134B+PBS组、敲减FAM134B+LPS组，图6F较图6D、6E凋亡细胞更多，图6D、6E、6F分别较图6A、6B、6C凋亡细胞更多
注：凋亡细胞呈现亮蓝色，活细胞呈现暗蓝色；2个空载体组转染空载慢病毒，2个敲减组转染含序列相似性家族134成员B（FAM134B）基因小干扰RNA序列的慢病毒
7 蛋白质印迹法检测的6组小鼠树突状细胞（DC）系DC2.4培养24 h凋亡相关蛋白的蛋白表达
注：caspase-3为胱天蛋白酶3，Bcl-2为B细胞淋巴瘤2，Bax为Bcl-2相关X蛋白；条带上方1、2、3、4、5、6分别指单纯磷酸盐缓冲液（PBS）组、单纯内毒素/脂多糖（LPS）组、空载体+PBS组、空载体+LPS组、敲减序列相似性家族134成员B（FAM134B）+PBS组、敲减FAM134B+LPS组；2个空载体组转染空载慢病毒，2个敲减组转染含FAM134B基因小干扰RNA序列的慢病毒
表1 5组小鼠树突状细胞（DC）系DC2.4中内质网自噬相关蛋白的蛋白表达比较（
表1. The comparison of protein expressions of reticulophagy associated proteins of mouse dendritic cell (DC) line DC2.4 in five groups
组别 样本数 FAM134B SEC61B LC3B-Ⅱ/LC3B-Ⅰ比值 LPS刺激0 h（不刺激）组 3 0.51±0.10 1.67±0.05 0.79±0.26 LPS刺激6 h组 3 0.75±0.30 1.11±0.21 0.91±0.18 LPS刺激12 h组 3 1.00±0.16 0.94±0.13 0.94±0.09 LPS刺激24 h组 3 1.20±0.07 0.83±0.22 1.59±0.09 LPS刺激72 h组 3 0.80±0.08 1.22±0.26 1.29±0.08 F值 7.70 8.93 13.13 P值 <0.001 <0.001 <0.001 P 1值 0.099 0.004 0.371 P 2值 0.004 0.001 0.289 P 3值 <0.001 <0.001 <0.001 P 4值 0.530 0.016 0.003 注：LPS为内毒素/脂多糖，FAM134B为序列相似性家族134成员B，LC3B为微管相关蛋白1轻链3B； F值、 P值为组间各指标总体比较所得； P 1值、 P 2值、 P 3值、 P 4值分别为LPS刺激6 h组、LPS刺激12 h组、LPS刺激24 h组、LPS刺激72 h组与LPS刺激0 h组各指标比较所得 
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表2 6组小鼠树突状细胞（DC）系DC2.4培养24 h凋亡相关蛋白的蛋白表达比较（
表2. Comparison of protein expressions of apoptosis‑related proteins of mouse dendritic cell (DC) line DC2.4 in six groups after 24 hours of culture
组别 样本数 剪切型caspase-3 Bax/Bcl-2比值 单纯PBS组 5 0.37±0.04 0.70±0.26 单纯LPS组 5 0.60±0.08 1.45±0.18 空载体+PBS组 5 0.37±0.11 1.18±0.12 空载体+LPS组 5 0.62±0.07 2.35±0.82 敲减FAM134B+PBS组 5 0.62±0.30 2.32±0.79 敲减FAM134B+LPS组 5 1.29±0.18 14.10±0.47 P 1值 0.032 0.033 P 2值 0.021 0.002 P 3值 <0.001 <0.001 P 4值 <0.001 <0.001 P 5值 <0.001 <0.001 注：PBS为磷酸盐缓冲液，LPS为内毒素/脂多糖，FAM134B为序列相似性家族134成员B，caspase-3为胱天蛋白酶3，Bcl-2为细胞淋巴瘤2，Bax为Bcl-2相关X蛋白；2个空载体组转染空载慢病毒，2个敲减组转染含 FAM134B基因小干扰RNA序列的慢病毒；剪切型caspase-3、Bax/Bcl-2比值的刺激类型的主效应， F值分别为44.27、575.43， P值均<0.001；细胞转染类型的主效应， F值分别为28.98、569.95， P值均<0.001；二者交互作用， F值分别为6.41、358.95， P值分别为0.006、<0.001； P 1值、 P 2值、 P 3值分别为单纯LPS组、空载体+LPS组、敲减FAM134B+LPS组与对应PBS处理组即单纯PBS组、空载体+PBS组、敲减FAM134B+PBS组各指标比较所得； P 4值、 P 5值分别为单纯LPS组、空载体+LPS组与敲减FAM134B+LPS组各指标比较所得
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