基于蛋白质组学探讨Ppp3cb和Ppm1g在NHE1基因敲除模型鼠海马组织中的表达  

Expression of Ppp3cb and Ppm1g in the hippocampus of NHE1 gene knockout rats based on proteomics

作　　者：马鹏飞 杨攀 郑乾 张祥明 屠秋霞 张春林 叶兰 冯占辉 MA Pengfei;YANG Pan;ZHENG Qian;ZHANG Xiangming;TU Qiuxia;ZHANG Chunlin;YE Lan;FENG Zhanhui(School of Clinical Medicine,Guizhou Medical University,Guiyang,Guizhou Province,550000;Research Center of Molecular Medical Engineering,Basic Medical School,Guizhou Medical University,Guiyang,Guizhou Province,550025,China;Department of Biology,Basic Medical School,Guizhou Medical University,Guiyang,Guizhou Province,550025,China)

机构地区：[1]贵州医科大学临床医学院,贵阳550000 [2]贵州医科大学基础医学院:分子医学工程研究中心,贵阳550025 [3]贵州医科大学基础医学院生物教研室,贵阳550025

出　　处：《陆军军医大学学报》2024年第11期1244-1253,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(8186050199,8236050283)。

摘　　要：目的通过NHE1基因敲除模型鼠的海马组织差异蛋白质组学分析,发现并明确Ppp3cb和Ppm1g的表达特征。方法①选取6只2周龄NHE1基因敲除模型鼠作为模型组,同周龄野生型小鼠6只作为对照组,采用琼脂糖凝胶电泳检测其基因型;应用旷场实验和强迫游泳实验对模型组和对照组小鼠进行行为学评估,并按照Racine评分标准对模型鼠进行癫痫发作分级;②通过串联质谱分析技术对模型组和对照组的海马组织进行差异蛋白筛选,基因本体论(Gene Ontology Analysis,GO)分析差异蛋白并进行注释和富集,蛋白网络数据库(search tool for the retrieval of interesting genes,STRING)分析差异蛋白之间的蛋白相互作用(protein-protein interaction,PPI);③应用qPCR和Western blot检测Ppp3cb和Ppm1g的转录和翻译水平,应用免疫组织化学技术分别观察其在组织中的表达量。结果①模型组小鼠NHE1基因未见表达,旷场实验中模型鼠的运动总距离较对照组减少(P=0.0073),跨越的格子数比对照组显著减少(P<0.0001)。强迫游泳实验结果显示,模型鼠不动的时间明显延长(P<0.0001);②以表达倍数(FC)≥1.2倍且P<0.05为筛选标准,检测到海马组织中845个差异表达的蛋白质点,其中有9个蛋白表达上调,7个蛋白表达下调。其中Ppp3cb下调,Ppm1g上调。GO功能注释结果表明,NHE1敲除后,分子功能(MF)富集在蛋白丝氨酸/苏氨酸磷酸酶活性的差异最显著,细胞成分(CC)富集在质膜部分的差异蛋白数量最多,生物过程(BP)富集在负向调节生物过程、免疫系统过程的差异蛋白数量最多。STRING分析显示差异蛋白Ppp3cb和Slc9a1直接作用,Ppm1g通过Ppp3cb和Slc9a1间接作用,Ppp3cb和Ppm1g之间相互作用。③Ppp3cb的转录和翻译水平减少,在组织中的表达量下降,而Ppm1g转录和翻译水平增加,在组织中的表达量上升(P<0.05)。结论本研究确定了NHE1基因敲除小鼠海马组织中差异蛋白Ppp3cb表达下调,而PpmObjective To investigate and validate the expression profiles of Ppp3cb and Ppm1g through differential proteomic analysis of hippocampal tissue in NHE1 gene knockout mice with proteomic analysis.Method①Six 2-week-old NHE1 knockout mice were selected as the model group,and 6 wild-type mice of the same age served as the control group,and their genotypes were detected by agar-gel electrophoresis.Open field test and forced swimming test were used to evaluate the behaviors of mice in the model group and control group,and epileptic seizure was graded according to Racine scoring.②Tandem mass spectrometry was employed to screen the differential proteins in the hippocampus tissues from the model group and the control group.Then the obtained differential proteins were annotated and enriched in the Gene Ontology(GO)database.Search tool for the retrieval of interesting genes(STRING)database was used to analyze protein-protein interaction(PPI)among different proteins.③The transcriptional and translational levels of Ppp3cb and Ppm1g were detected by qPCR and Western blotting,respectively,and their expression levels in the tissues were observed with immunohistochemistry.Results①NHE1 was not expressed in the model group.The mice of the model group had shorter total movement distance(P=0.0073)and less crossing cells(P<0.0001)in open field test,and longer period of immobility in forced swimming test(P<0.0001)when compared with those from the control group.②When fold change≥1.2 times and P<0.05 were set as the significant threshold for differential expression,845 differentially expressed protein sites were detected in the hippocampus,among which 9 proteins(including Ppm1g)were up-regulated and 7 ones(including Ppp3cb)were down-regulated.Gene Ontology(GO)functional analysis showed that after NHE1 knockout,the most significant differences were observed in the concentration of molecular function(MF)related to protein serine/threonine phosphatase activity,concentration of cellular component(CC)related to the plasma membrane

关 键 词：NHE1基因敲除 癫痫 海马 蛋白质组学 Ppp3cb Ppm1g 

分 类 号：R338.26[医药卫生—人体生理学] R394-33[医药卫生—基础医学] R394.2