凋亡相关斑点样蛋白寡聚阻断剂对小鼠脊髓损伤急性期局部基因转录表达影响的转录组分析  

CRID3,a blocker of apoptosis-associated speck-like protein containing a CARD,influences local gene transcription in mice with acute spinal cord injury:a transcriptomic analysis

作　　者：李璟璐 王赛男 王阳阳 付贵强 王颖 胡建国[1,2] 唐洁 吕合作[1,2,3] Li Jinglu;Wang Sainan;Wang Yangyang;Fu Guiqiang;Wang Ying;Hu Jianguo;Tang Jie;Lyu Hezuo(Clinical Laboratory,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,Anhui Province,China;Key Laboratory of Tissue Transplantation,Bengbu Medical College,Bengbu 233000,Anhui Province,China;Department of Immunology,Bengbu Medical College,Bengbu 233000,Anhui Province,China)

机构地区：[1]蚌埠医学院第一附属医院检验科,安徽蚌埠233004 [2]蚌埠医学院组织移植安徽省重点实验室,安徽蚌埠233000 [3]蚌埠医学院免疫学教研室,安徽蚌埠233000

出　　处：《中国组织工程研究》2022年第23期3620-3632,共13页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81772321),项目负责人:吕合作;蚌埠医学院首批“512人才培养计划”项目(51201109),项目负责人:吕合作。

摘　　要：背景:炎症小体在脊髓损伤后发挥重要作用,凋亡相关斑点样蛋白是炎症小体的共同接头蛋白。作者前期的研究表明,一种特异性的凋亡相关斑点样蛋白寡聚阻断剂CRID3,可以通过抑制凋亡相关斑点样蛋白寡聚化而抑制炎症小体的活化和相应细胞因子的产生,进而改善损伤脊髓局部微环境,发挥神经保护作用。但是其在转录水平对损伤脊髓的影响尚未见报道。目的:采用转录组测序技术分析CRID3对小鼠脊髓损伤急性期(8 h)局部基因转录表达的影响。方法:共取雌性8周龄C57BL/6小鼠30只,体质量18-20 g,随机分为假手术组和脊髓损伤组,将脊髓损伤后的小鼠分为模型对照组和给药组,给药组术后腹腔注射CRID3(50 mg/kg),对照组只注射等体积的生理盐水。脊髓损伤后8 h,每组各灌注取材3只小鼠,取脊髓冰冻切片进行苏木精-伊红染色,确定损伤模型是否成功。损伤后8 h每组各取3只小鼠,取脊髓提取纯化总RNA,进行文库制备和转录组测序,用DESeq2软件分析3组模型的差异基因表达。同转录组测序,模型对照组和给药组各取6只小鼠脊髓提取纯化总RNA,采用反转录实时定量PCR方法验证转录组测序结果。用GOseq R软件和KOBAS软件对差异基因分别进行基因本体分析和京都基因与基因组百科全书富集分析;结合文献挖掘与炎症小体相关的信号通路、深入分析CRID3对其相关基因表达的影响;与CRID3作用相关的基因表达蛋白String互作分析。结果与结论:①苏木精-伊红染色结果表明脊髓损伤模型构建成功;②转录组测序结果分析显示,与假手术组比较,模型对照组有5661个差异表达基因,其中包括3427个上调和2224个下调基因;与模型对照组比较,给药组有2924个差异表达基因,其中包含1409个上调基因和1515个下调基因;③基因本体分析结果表明,差异表达的基因主要富含趋化因子受体结合、G-蛋白偶联受体结合、BACKGROUND:Inflammasomes play an important role in spinal cord injury.Apoptosis-associated speck-like protein containing a CARD is a common adaptor protein of inflammasome.Our previous studies have shown that CRID3,a specific oligomerization blocker of apoptosis-associated speck-like protein containing a card,can inhibit the activation of inflammasome and the production of corresponding cytokines by inhibiting the oligomerization of apoptosis-associated speck-like protein containing a card,so as to improve the local microenvironment of the injured spinal cord and play a neuroprotective role.However,its effect on spinal cord injury at transcriptional level has not been reported.OBJECTIVE:To investigate the effect of CRID3 on local gene transcription at acute phase(8 hours)of spinal cord injury in mice by using RNA-sequencing.METHODS:Thirty female C57BL/6 mice aged 8 weeks and weighing 18-20 g were divided into a sham operation group and a spinal cord injury group.Mice in the spinal cord injury group were randomly subdivided into a control group and a CRID3 administration group.Mice in the CRID3 administration group were intraperitoneally injected with CRID3(50 mg/kg)after operation,while mice in the control group were injected with an equal volume of physiological saline solution.At 8 hours after spinal cord injury,three mice from each group were perfused,and the spinal cord was taken to make frozen sections that were then stained with hematoxylin-eosin to determine whether the spinal cord injury model was successfully established.Meanwhile,another three mice were selected from each group to taken spinal cord tissue.The total RNA was then extracted and purified for library preparation and RNA-sequencing.The differential gene expression of the three groups was analyzed by DESeq2 software.Another six mice were selected from the control group and the CRID3 administration group,and spinal cord tissue was taken to extract and purify total RNA.The results of RNA-sequencing were verified by real-time quantitative reverse

关 键 词：脊髓损伤 CRID3 小鼠 转录组测序 炎症小体 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]