【摘要】 目的 基于串联质谱标记相对定量蛋白质组学技术联合转录组学探讨肌球蛋白轻链-2（Myosin light chain-2,Myl2）和肌钙蛋白I3（Troponin I3,Tnni3）在二乙酰吗啡致心肌细胞节律异常中的作用。方法 培养原代SD大鼠乳鼠心肌细胞，采用免疫荧光法鉴定原代SD大鼠乳鼠心肌细胞。将心肌细胞分为CON组和Drug组(10^-4 mol/L二乙酰吗啡),分别采用串联质谱标记相对定量蛋白质组学和转录组学方法获取两组的差异表达基因，利用韦恩图取交集，对筛选出的共有差异表达基因进行基因本体论（Gene ontology, GO）功能和基因组百科全书（Kyoto encyclopedia of genes and genomes, KEGG）通路富集分析，利用网络拓扑结构中的各项参数，根据基因相似性筛选核心差异表达基因。采用Fluo-3 AM探针检测CON组和Drug组细胞内[Ca²⁺]i水平，采用蛋白免疫印迹法检测CON组和Drug组Myl2、Tnni3蛋白的表达变化。结果 原代SD大鼠乳鼠心肌细胞纯度在95%以上，串联质谱标记相对定量蛋白质组学和转录组学筛选出的共有差异表达基因为109个。GO功能富集分析显示，共有差异表达基因的功能主要有代谢、细胞杀伤和转录调节等，KEGG通路富集分析显示，共有差异表达基因主要富集于心肌收缩信号通路、钙信号通路等；根据基因相似度均值进行排列，排名前3的分别为Tnni3、Tnnt2、Myl2。与CON组相比，Drug组细胞内[Ca²⁺]i升高，Myl2和Tnni3蛋白表达水平升高（P<0.05）。结论 Myl2和Tnni3参与二乙酰吗啡致心肌细胞节律异常过程。更多还原

【Abstract】 Objective To investigate the role of myosin light chain-2（Myl2） and troponin I3（Tnni3） in diacetylmorphine-induced cardiac rhythm abnormalities in cardiomyocytes by combining tandem mass spectrometry labeling of relative quantitative proteomics techniques and transcriptomics. Methods Primary SD rat neonatal cardiomyocytes were cultured and identified by immunofluorescence. The cardiomyocytes were divided into CON group and Drug group(10^-4 mol/L diacetylmorphine). Differentially expressed genes were obtained using tandem mass spectrometry labeling of relative quantitative proteomics and transcriptomics, respectively. Venn diagram was used to obtain overlapping genes, the selected common differentially expressed genes were subjected to gene ontology（GO） functional and kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis. Utilizing various parameters in network topology, core differentially expressed genes were screened based on gene similarity. Intracellular [Ca²⁺]i levels in the CON and Drug groups were detected using Fluo-3 AM probe, and Western blot was used to determine the protein expression of Myl2 and Tnni3. Results The purity of primary SD rat neonatal cardiomyocytes was over 95%. A total of 109 shared differentially expressed genes were identified by tandem mass spectrometry labeling of relative quantitative proteomics and transcriptomics. GO functional enrichment analysis revealed that the functions of the shared differentially expressed genes mainly involved metabolism, cell killing, and transcriptional regulation. KEGG pathway enrichment analysis indicated that the shared differentially expressed genes were mainly enriched in cardiac muscle contraction signaling pathway and calcium signaling pathway. Ranked according to the average gene similarity, the top three are Tnni3, Tnnt2 and Myl2. Compared with the CON group, intracellular [Ca²⁺]i levels increased in the Drug group, and the expression levels of Myl2 and Tnni3 proteins were elevated（P<0.05）. Conclusion Myl2 and Tnni3 are involved in the process of diacetylmorphine-induced cardiac rhythm abnormalities in cardiomyocytes.更多还原