目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing fac...目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing factor,AIF)。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合,变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平,过表达FBXO22可下调AIF水平。流式细胞术结果显示,敲低FBXO22可促进由甲基硝基亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)等诱导的结肠癌细胞凋亡,过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。展开更多
目的·分析Fbxo22基因敲除小鼠的表型,为探索FBXO22的生物学功能提供理论依据。方法·利用CRISPR-Cas9(clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9)技术成功构建Fbxo22全身敲除小...目的·分析Fbxo22基因敲除小鼠的表型,为探索FBXO22的生物学功能提供理论依据。方法·利用CRISPR-Cas9(clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9)技术成功构建Fbxo22全身敲除小鼠,观察胚胎和小鼠的外观,测定其数量和质量,并分析小鼠的进食量和存活时间。结果·Fbxo22敲除小鼠胚胎期17.5/18.5 d的胚胎数量符合孟德尔遗传定律,外观未见异常,但是多数Fbxo22敲除小鼠在出生后48 h内死亡。少数存活小鼠体型偏小,进食减少,存活时间缩短。结论·FBXO22对于小鼠出生后早期存活和正常发育有重要作用。展开更多
OBJECTIVE Social anxiety disorder,also known as social phobia,is the most common of all anxiety disorders.Despite the seriousness of this disorder,it has rarely been studied and as a consequence,little is known about ...OBJECTIVE Social anxiety disorder,also known as social phobia,is the most common of all anxiety disorders.Despite the seriousness of this disorder,it has rarely been studied and as a consequence,little is known about its underlying neurobiology.Growing evidence suggests that inhibitory neurons dysfunction could affect the balance of neuronal activity in psychotic disorders including anxiety,SAD,etc.Ephrin-B(EB) proteins were previously demonstrated to ensure normal distribution and function of inhibitory interneurons in the brain.We thus proposed that deletion of EB2 in inhibitory neurons might destroy the homeostasis of excitatory neurons and inhibitory neurons,and induce behavioral deficits associated with psychotic diseases.METHODS(1)Mice.We generated conditional EB2 knockout mice as a model of SAD and characterized the behaviors and the biochemical changes in the brains of the knockout mice.We also generated a mouse line with a selective deletion of EB2 from vGAT neurons by breeding a v GAT-Cre line with a loxP-flanked alleles in EB2.For EB2 detection,we crossed vGAT-Cre;EphrinB2 loxp/loxp mice with Rosa26-STOP-tdTomato Cre indicator(Ai9) mice to label the vGAT neurons with tdTomato.Mice were maintained in a mixed CD1/129 genetic background.Al experiments involving mice were carried out in accordance with the Shanghai Jiaotong University.Genotypes were unlocked only after completion of analysis.(2) General procedures for behavioral testing.Open field test:Locomotor activity was measured using an open field test.The size of the open field box was 44 cm×44 cm×44 cm.Each mouse was placed in the corner of the open-field apparatus at the beginning of the test,and allowed to explore it for 15 min.The total distance traveled(in cm) and counts for stereotyped behaviors were recorded and analyzed.The total distance traveled was used as an index of locomotor activity.The percentage of center zone entries was considered as anxiety indexes.EPM test:The elevated plus maze apparatus was made of dark gray plastic and comprised two open arms(30 cm×7 cm×0.25 cm) opposed to two enclosed arms(30 cm × 7 cm × 15 cm) elevated60 cm from the floor.Animals were placed in the central area of the apparatus with their head facing an enclosed arm(test duration,5 min).Digitized video recordings with EthoVision software were used for behavioral analysis.The percentage of time spent in open arms and the percentage of open-arm entries were calculated.Social behaviors:Briefly,a top open acrylic box was divided into three compartments by two clear acrylic glass partitions with an opening in the bottom.Wire mesh cages that are either empty or have social stimulus mouse in it were put in the center of each outer compartment.Unfamiliar naive CD1/129 mice of the same age were used as stimulus mice.Prior to the test,the mouse was introduced into the center chamber for 5 min,with both gates closed.Gates were then open and the test mouse was acclimated to explore the empty three-chambered apparatus freely for another 10 min.After the initial 15 min habituation session,mice were placed in the center compartment and allowed to explore freely all the three compartments.To examine the social contact,the following activities were recorded via a video surveillance for 10 min.The number of entries to each compartment,the time spent in each compartment as well as the time and frequency each mouse spent in sniffing were analyzed with EthoVision XT 8.5(Noldus,Wageningen,Netherlands).RESULTS(1)Social preference assays were performed in WT-vGATCre,EB2-flox,and EB2-vGATCre mice respectively.To evaluate social approach behavior,we used a three-chamber social arena to evaluate animals for their social interaction.The control groups of WTvGATCre,EB2-flox mice showed a significant preference for spending time in the social chamber(P<0.05);whereas the EB2-vGATCre mice did not show this preference and spent roughly equal time in the social and nonsocial chambers.(2)To determine if the decrease in social preference could result from changes in locomotion,we compared locomotor activity in EB2-vG ATCre mice and control mice.We did not observe a significant lower locomotive activity in mice.Hence,EB2 deletion caused a change in social preference but not in locomotion.Taken together,EB2 deletion exerts abnormal psychotogenic activity of social deficits which was often considered as an important feature of SAD.(3) The time spent in the open arms is widely used to measure anxiety in mice,with greater time spent in the open arm being considered as less anxious behavior.To normalize for individual differences in overall activity level,the time spent in the open arms was divided by the total time.The ratio was higher for WT-vGATCre mice and EB2-loxp mice as compared to the EB2-vG ATCre mice(P=0.0112,P=0.0197).Thus,results showed that there was a change in anxiety levels from in EB2-vGATCre mice as compared with control mice.CONCLUSION EB2 may be a candidate risk gene for SAD and that the EB2 conditional knockout model could be a tool for studying the underlying mechanisms in SAD.展开更多
文摘目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing factor,AIF)。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合,变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平,过表达FBXO22可下调AIF水平。流式细胞术结果显示,敲低FBXO22可促进由甲基硝基亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)等诱导的结肠癌细胞凋亡,过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。
基金National Natural Science Foundation of China (8150115381571326).
文摘OBJECTIVE Social anxiety disorder,also known as social phobia,is the most common of all anxiety disorders.Despite the seriousness of this disorder,it has rarely been studied and as a consequence,little is known about its underlying neurobiology.Growing evidence suggests that inhibitory neurons dysfunction could affect the balance of neuronal activity in psychotic disorders including anxiety,SAD,etc.Ephrin-B(EB) proteins were previously demonstrated to ensure normal distribution and function of inhibitory interneurons in the brain.We thus proposed that deletion of EB2 in inhibitory neurons might destroy the homeostasis of excitatory neurons and inhibitory neurons,and induce behavioral deficits associated with psychotic diseases.METHODS(1)Mice.We generated conditional EB2 knockout mice as a model of SAD and characterized the behaviors and the biochemical changes in the brains of the knockout mice.We also generated a mouse line with a selective deletion of EB2 from vGAT neurons by breeding a v GAT-Cre line with a loxP-flanked alleles in EB2.For EB2 detection,we crossed vGAT-Cre;EphrinB2 loxp/loxp mice with Rosa26-STOP-tdTomato Cre indicator(Ai9) mice to label the vGAT neurons with tdTomato.Mice were maintained in a mixed CD1/129 genetic background.Al experiments involving mice were carried out in accordance with the Shanghai Jiaotong University.Genotypes were unlocked only after completion of analysis.(2) General procedures for behavioral testing.Open field test:Locomotor activity was measured using an open field test.The size of the open field box was 44 cm×44 cm×44 cm.Each mouse was placed in the corner of the open-field apparatus at the beginning of the test,and allowed to explore it for 15 min.The total distance traveled(in cm) and counts for stereotyped behaviors were recorded and analyzed.The total distance traveled was used as an index of locomotor activity.The percentage of center zone entries was considered as anxiety indexes.EPM test:The elevated plus maze apparatus was made of dark gray plastic and comprised two open arms(30 cm×7 cm×0.25 cm) opposed to two enclosed arms(30 cm × 7 cm × 15 cm) elevated60 cm from the floor.Animals were placed in the central area of the apparatus with their head facing an enclosed arm(test duration,5 min).Digitized video recordings with EthoVision software were used for behavioral analysis.The percentage of time spent in open arms and the percentage of open-arm entries were calculated.Social behaviors:Briefly,a top open acrylic box was divided into three compartments by two clear acrylic glass partitions with an opening in the bottom.Wire mesh cages that are either empty or have social stimulus mouse in it were put in the center of each outer compartment.Unfamiliar naive CD1/129 mice of the same age were used as stimulus mice.Prior to the test,the mouse was introduced into the center chamber for 5 min,with both gates closed.Gates were then open and the test mouse was acclimated to explore the empty three-chambered apparatus freely for another 10 min.After the initial 15 min habituation session,mice were placed in the center compartment and allowed to explore freely all the three compartments.To examine the social contact,the following activities were recorded via a video surveillance for 10 min.The number of entries to each compartment,the time spent in each compartment as well as the time and frequency each mouse spent in sniffing were analyzed with EthoVision XT 8.5(Noldus,Wageningen,Netherlands).RESULTS(1)Social preference assays were performed in WT-vGATCre,EB2-flox,and EB2-vGATCre mice respectively.To evaluate social approach behavior,we used a three-chamber social arena to evaluate animals for their social interaction.The control groups of WTvGATCre,EB2-flox mice showed a significant preference for spending time in the social chamber(P<0.05);whereas the EB2-vGATCre mice did not show this preference and spent roughly equal time in the social and nonsocial chambers.(2)To determine if the decrease in social preference could result from changes in locomotion,we compared locomotor activity in EB2-vG ATCre mice and control mice.We did not observe a significant lower locomotive activity in mice.Hence,EB2 deletion caused a change in social preference but not in locomotion.Taken together,EB2 deletion exerts abnormal psychotogenic activity of social deficits which was often considered as an important feature of SAD.(3) The time spent in the open arms is widely used to measure anxiety in mice,with greater time spent in the open arm being considered as less anxious behavior.To normalize for individual differences in overall activity level,the time spent in the open arms was divided by the total time.The ratio was higher for WT-vGATCre mice and EB2-loxp mice as compared to the EB2-vG ATCre mice(P=0.0112,P=0.0197).Thus,results showed that there was a change in anxiety levels from in EB2-vGATCre mice as compared with control mice.CONCLUSION EB2 may be a candidate risk gene for SAD and that the EB2 conditional knockout model could be a tool for studying the underlying mechanisms in SAD.
