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· 18 · 南 京 医 科 大 学 学 报 2023年1月

final concentration of 300 μmol/L for 6 h，and then randomly divided into control group and caspase⁃3 inhibitor group（Ac⁃DEVD⁃CHO，
15 μmol/L）. The sequencing was completed by using the Illumina Hiseq 2500 platform. Enrichment analysis and an interaction
network were performed to screen differential genes. Quantitative PCR was used to verify the first 10 differentially expressed genes
（DEGs）. The HK⁃2 cells treated with H 2O2 （300 μmol/L）for 6 h were then divided into control group，caspase⁃3 inhibitor group
（Ac⁃DEVD⁃CHO，15 μmol/L），CTNNB1 group（pcDNA3.1（+）⁃CTNNB1），caspase⁃3 inhibitor+CTNNB1 group（Ac⁃DEVD⁃CHO，
15 μmol/L+pcDNA3.1（+）⁃CTNNB1）and caspase⁃3 inhibitor+CTNNB1 NC group（Ac⁃DEVD⁃CHO，15 μmol/L+pcDNA3.1（+）⁃
CTNNB1 NC）randomly. MTT assay was used to detect cell proliferation，flow cytometry was used to detect cell apoptosis and ROS
level，Western blot was used to detect apoptosis⁃related proteins. Results：A total of 185 DEGs were selected in the control group and
the caspase⁃3 inhibitor group. Quantitative PCR showed FIS1，EZR，COL7A1，RPL5，MAP4，CEBPB and CTNNB1 mRNA were lowly
expressed（all P < 0.05）and SNRPB mRNA was highly expressed in caspase⁃3 inhibitor group（P < 0.05）. The proliferation was higher
in the caspase⁃3 inhibitor group and was lower the CTNNB1 group compared to the control（both P < 0.05）. The proliferation of the
caspase⁃3 inhibitor+CTNNB1 group was lower than that of the caspase⁃3 inhibitor group（P < 0.05），but still higher than the control
（P < 0.05）. The apoptosis and the expression of cleaved⁃caspase⁃3 and cleaved⁃PARP were lower in caspase⁃3 inhibitor group and
were higher in CTNNB1 group compared to the control（both P < 0.05）. The apoptosis and the expression of cleaved⁃caspase⁃3 and
cleaved⁃PARP in caspase⁃3 inhibitor+CTNNB1 group were higher than those in the caspase⁃3 inhibitor group（P < 0.05），but were still
lower than the control（P < 0.05）. The ROS in the caspase⁃3 inhibitor group was lower but was higher in CTNNB1 group compared to
that of the control group（P < 0.05）. The ROS in the caspase⁃3 inhibitor+CTNNB1 group was higher than that of the caspase⁃3 inhibitor
group（P < 0.05），but was still lower than the control（P < 0.05）. Conclusion：Aberrant gene expression induced by caspase⁃3 inhibitor
is associated with renal injury under oxidative stress. The injury of oxidative stress on renal tubular epithelial cells is caspase ⁃ 3
dependent. Inhibition of caspase⁃3 has a protective effect on renal injury and CTNNB1 is involved in this process.
［Key words］ kidney injury；oxidative stress；mitochondria；bioinformatics；CTNNB1
［J Nanjing Med Univ，2023，43（01）：017⁃026］

氧化应激损伤是造成急性和慢性肾损伤的重 细胞中超氧阴离子浓度的调节。但是目前对肾小
要机制之一。人体正常的生物代谢会产生超氧阴 管上皮损伤中 ROS 和 caspase⁃3 途径细胞凋亡的研
离子自由基、过氧化氢等活性氧（reactive oxygen 究还较少，caspase⁃3 对 ROS 的调控是直接起作用，
species，ROS），体内完善的抗 ROS 系统能及时将多 还是引起了其他基因、蛋白或通路的变化？除了线
余的ROS清除，较低水平的ROS对肾小管和微循环 粒体⁃caspase 途径介导细胞凋亡，其他凋亡通路是
［1］
的生理调节有积极作用 ；但是在受到各种病原体 否也参与其中？这些问题目前尚不清楚。
或炎症因子刺激时，肾小管细胞中氧化还原状态调 本研究拟利用芯片测序，对caspase⁃3抑制剂处
控失衡，造成 ROS 在肾组织蓄积、细胞膜损伤和超 理的氧化应激状态下肾小管上皮细胞转录组进行
［2］
氧阴离子渗漏，最终导致肾损伤 。以氧化应激为 分析，筛选 caspase⁃3 抑制剂引起的差异表达基因
治疗靶点已经成为肾损伤治疗的热点。 （differentially expressed gene，DEG），对DEG进行GO
Caspase⁃3 介导的线粒体途径凋亡是经典的细 分析和 KEGG 富集分析，对筛选出的关键基因进行
胞凋亡通路。有研究指出，氧化应激损伤和caspase⁃3 生物学验证，深入探讨和揭示caspase⁃3调控ROS损
介导的线粒体途径细胞凋亡存在关联 ［3-4］ 。我们在 伤肾小管上皮细胞的机制。
前期研究中发现，H2O2 处理的人肾小管上皮细胞
1 材料和方法
HK⁃2 中氧化应激水平升高，细胞增殖受到抑制、细
胞凋亡增多；而caspase⁃3抑制剂能降低H2O2处理后 1.1 材料
肾小管细胞ROS水平，对氧化应激引起的细胞损伤 人肾小管上皮细胞HK⁃2（上海中科院典型培养
有改善作用，说明肾小管上皮细胞的氧化应激损伤 物保藏中心）。Ac⁃DEVD⁃CHO（上海碧云天生物技
具有caspase⁃3依赖性，抑制caspase⁃3途径的细胞凋 术有限公司）、VigoFect 转染试剂盒（北京威格拉斯
亡对 ROS 引起的脓毒症肾小管上皮细胞损伤有一 生物技术有限公司）、TRIzol 试剂盒（TaKaRa 公司，
定保护作用 。Kaleem 等 指出，caspase 的释放受 日本）、SuperReal PreMix Plus（北京天根生化科技有
［6］
［5］