山东大学学报 (医学版) ›› 2023, Vol. 61 ›› Issue (9): 57-68.doi: 10.6040/j.issn.1671-7554.0.2023.0427
高玉杰,龙启福,胡英,许玉珍,王茹,永胜
GAO Yujie, LONG Qifu, HU Ying, XU Yuzhen, WANG Ru, YONG Sheng
摘要: 目的 利用生物信息学分析挖掘低氧介导线粒体损伤的关键基因,并分析关键基因与小鼠肾脏组织凋亡的相关性,进一步阐明高原低氧条件下小鼠肾脏组织损伤的分子作用机制。 方法 构建30 d常氧与低氧动物模型,分别作为平原常氧组(n=5)和高原低氧组(n=5),测定血气分析指标与小鼠肾脏指数,通过H&E染色观察小鼠肾脏组织病理学改变,对平原常氧组与高原低氧组小鼠肾脏组织进行转录组学测序。随后将测序得到的差异表达基因(DEGs)分别与线粒体数据库、凋亡相关基因数据库进行关联,鉴定差异线粒体功能相关基因(DE-MFRGs)与差异凋亡相关基因(DE-ARGs),蛋白质互作(PPI)网络筛选Hub基因,利用生物信息学对DEGs、DE-MFRGs、DE-ARGs进行富集分析,并通过RT-qPCR 和 Western blotting对Hub 基因及凋亡基因进行验证。 结果 与平原常氧组相比,高原低氧组小鼠肾脏指数显著下降。H&E染色结果显示,高原低氧组小鼠肾小球明显萎缩,肾小管上皮细胞肿胀、破裂,炎细胞浸润,部分肾小管上皮细胞体积增大,胞浆疏松淡染,可见空泡变性。低氧暴露条件下共鉴定出3 007个DEGs,其中有464个基因与线粒体功能相关,被定义为DE-MFRGs。对DE-MFRGs进行KEGG富集分析发现,其主要富集在帕金森病信号通路、氧化磷酸化信号通路、产热信号通路、阿尔兹海默病信号通路、三羧酸循环信号通路、NAFLD信号通路、代谢信号通路等与线粒体功能相关的通路中。通过构建PPI网络共鉴定出3个Hub基因(SOD2、TUFM、MRPL12),SOD2、TUFM、MRPL12的mRNA和蛋白表达量均下调。进一步发现Hub基因与33个ARGs基因均出现正相关与负相关,并验证了小鼠肾脏组织ARGs中CASPASE3、BCL2、BAK基因的mRNA与蛋白表达量,结果显示,CASPASE3与BAK基因表达上调,BCL2基因表达下调。 结论 高原低氧环境通过调控SOD2、TUFM与MRPL12基因的表达,介导小鼠肾脏组织线粒体功能紊乱,进一步诱导小鼠肾脏组织损伤与凋亡。
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