低氧对牛肾细胞增殖和线粒体自噬的影响

doi:10.3864/j.issn.0578-1752.2026.06.014

摘要/Abstract

摘要：

【背景】低氧是一种独特的环境应激，广泛地影响着呼吸、循环、泌尿、消化等多个系统的生理功能，在个体、组织器官以及细胞水平产生不同程度的影响，严重时引起机体组织器官损伤和疾病，在细胞水平涉及形态结构、代谢、增殖、自噬及凋亡等多个方面。肾脏是泌尿系统的重要组成部分，对于维持体内平衡、调节水电解质和酸碱平衡以及消除代谢废物至关重要，属于低氧敏感组织。目前，低氧对牛肾脏细胞的影响及其分子机制尚不明确。【目的】以牛肾细胞系（madin-darby bovine kidney，MDBK）为细胞模型，探讨不同低氧浓度（11% O₂、5% O₂和1% O₂）和不同低氧处理时间(24、48和72 h)对牛肾细胞的增殖能力、超微结构、线粒体功能及低氧应激能力的影响，为进一步探究牛肾脏低氧适应机制提供了试验依据，也为深入理解低氧条件下细胞的生存策略和线粒体自噬的调控机制提供基础。【方法】在体内，不同细胞和细胞器感受到的氧分压不同，在常氧下，氧分压为159.22 mmHg（20.95%），动脉氧分压为100 mmHg（13%），静脉氧分压（PaO₂）为40 mmHg（5.2%），线粒体氧分压为4—20 mmHg（0.52%—2.60%），鉴于此，本研究选择11% O₂、5% O₂和1% O₂浓度作为低氧处理条件。将牛肾细胞系MDBK接种于含10%胎牛血清的DMEM完全培养基中，在37 ℃、5% CO₂条件下培养至对数生长期，随后转移至三气培养箱，分别在11% O₂、5% O₂和1% O₂浓度下培养24、48和72 h，通过MTT法检测低氧对牛肾细胞增殖能力的影响，通过透射扫描电子显微镜观察低氧条件下MDBK细胞超微结构的变化，通过JC-1荧光探针法检测线粒体膜电位，通过DCFH-DA荧光探针法检测活性氧的产生，通过实时荧光定量PCR和Western Blot检测低氧相关基因以及自噬相关基因的表达情况。【结果】随着氧气浓度的下降和处理时间的延长，细胞增殖能力受到显著抑制，且氧气浓度越低处理时间越长抑制越显著；低氧导致线粒体结构损伤和功能障碍，表现为线粒体肿胀、嵴排列紊乱甚至消失，且线粒体数量也显著减少，部分线粒体发生固缩，出现线粒体被膜性结构包绕及线粒体自噬的现象，同时线粒体膜电位显著降低；细胞内活性氧水平显著升高；低氧相关基因EPAS1和PPARα的表达显著升高，线粒体自噬相关基因PINK1、PRKN、BNIP3和BNIP3L(NIX)的表达水平显著上调。【结论】5%和1%氧气浓度处理显著抑制牛肾细胞的增殖活力，并导致线粒体结构损伤和功能障碍，线粒体膜电位下降，活性氧产生增加。在应答低氧胁迫过程中，牛肾细胞通过激活线粒体自噬以清除功能失调的线粒体，从而减轻氧化应激，以维持细胞内环境的稳定。

关键词: 低氧, 牛肾细胞, 增殖, 线粒体自噬, 氧化应激

Abstract:

【Background】 Hypoxia is a unique environmental stressor that widely influences the physiological functions of multiple systems, including respiratory, circulatory, urinary, and digestive systems, exerting varying degrees of impact at the individual, organ, and cellular levels. Severe hypoxia can cause tissue and organ damage and disease, involving morphological structure, metabolism, proliferation, autophagy, and apoptosis at the cellular level. The kidney, a crucial component of the urinary system essential for maintaining homeostasis, regulating water-electrolyte and acid-base balance, and eliminating metabolic waste, is sensitive to hypoxia. Currently, the effects of hypoxia on bovine kidney cells and their underlying molecular mechanisms remain unclear. 【Objective】 This study employed the Madin-Darby Bovine Kidney (MDBK) cell line as a cellular model to investigate the effects of varying hypoxic concentrations (11% O₂, 5% O₂, and 1% O₂) and treatment durations (24, 48, and 72 h) on the proliferative capacity, ultrastructure, mitochondrial function, and hypoxic stress response of bovine kidney cells, in order to provide experimental evidence for further exploration of the hypoxic adaptation mechanisms in bovine kidney and a foundation for understanding cellular survival strategies and the regulatory mechanisms of mitophagy under hypoxic conditions. 【Method】 Different cells and organelles experience distinct oxygen partial pressures. Under normoxic conditions, atmospheric oxygen partial pressure was 159.22 mmHg (20.95%), arterial oxygen partial pressure was 100 mmHg (13%), venous oxygen partial pressure (PaO₂) was 40 mmHg (5.2%), and mitochondrial oxygen partial pressure ranges from 4-20 mmHg (0.52%-2.60%). Based on this, 11% O₂, 5% O₂, and 1% O₂ were selected as hypoxic treatment conditions. MDBK cells were seeded in DMEM complete medium supplemented with 10% fetal bovine serum and cultured at 37 ℃ with 5% CO₂ until reaching the logarithmic growth phase. Cells were then transferred to a tri-gas incubator and cultured under 11% O₂, 5% O₂, and 1% O₂ for 24, 48, and 72 h. The effects of hypoxia on bovine kidney cell proliferation were assessed by MTT assay. Ultrastructural changes in MDBK cells under hypoxic conditions were observed by transmission electron microscopy. Mitochondrial membrane potential was measured using the JC-1 fluorescent probe method. Reactive oxygen species (ROS) production was detected by the DCFH-DA fluorescent probe method. The expression of hypoxia-related and autophagy-related genes was analyzed by real-time quantitative PCR and Western blotting. 【Result】 Cell proliferation was significantly inhibited with decreasing oxygen concentration and prolonged treatment time, with more pronounced inhibition at lower oxygen concentrations and longer durations. Hypoxia caused mitochondrial structural damage and functional impairment, manifested as mitochondrial swelling, disorganized or absent cristae, a significant reduction in mitochondrial number, condensation of some mitochondria, and the presence of mitochondria enveloped by membranous structures indicative of mitophagy. Concurrently, mitochondrial membrane potential decreased significantly, but intracellular ROS levels increased markedly. The expression of hypoxia-related genes EPAS1 and PPARα was significantly upregulated, while the expression levels of mitophagy- related genes PINK1, PRKN, BNIP3, and BNIP3L (NIX) were significantly elevated. 【Conclusion】 This study demonstrated that treatment with 5% and 1% oxygen concentrations significantly inhibited the proliferative activity of bovine kidney cells, causing mitochondrial structural damage and dysfunction, decreased mitochondrial membrane potential, and increased ROS production. In response to hypoxic stress, bovine kidney cells activate mitophagy to eliminate dysfunctional mitochondria, thereby alleviating oxidative stress and maintaining intracellular homeostasis.

Key words: hypoxia, bovine renal cells, proliferation, mitophagy, oxidative stress

格日其木格, 普布占堆, 徐青, 侯玲玲. 低氧对牛肾细胞增殖和线粒体自噬的影响[J]. 中国农业科学, 2026, 59(6): 1333-1347.

GERIQIMUGE, PUBUZHANDUI, XU Qing, HOU LingLing. Effects of Hypoxia on Proliferation of Bovine Renal Cells and Mitochondrial Autophagy[J]. Scientia Agricultura Sinica, 2026, 59(6): 1333-1347.

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	10 cm皿 10 cm dish (cells/dish)	3.5 cm皿 3.5 cm dish (cells/dish)	6孔板 6-well plate (cells/well)	24孔板 24-well plate (cells/well)	96孔板 96-well plate (cells/well)
24 h	3×10⁶	4.8×10⁵	7.2×10⁵	/	7×10³
48 h	2×10⁶	2.8×10⁵	3.6×10⁵	/	5×10³
72 h	1.3×10⁶	1.6×10⁵	1.8×10⁵	4×10⁴	3×10³

成分 Component	使用量 Volume (μL)
2 × FastFire qPCR PreMix	5
正向引物（10 μmol·L^-1）Primer-Forward（10μmol·L^-1）	0.3
反向引物（10μmol·L^-1）Primer-Reverse（10μmol·L^-1）	0.3
cDNA模板 cDNA template	1
RNase-Free H₂O	3.4
合计 Total	10

温度 Temperature (℃)	时间 Time	循环数 Cycle number
95	1 min	1
95	5 s	/
60	30 s	40

基因 Gene	引物序列 Primer sequences (5'→3')	产物大小 Product size (bp)	退火温度 Annealing temperature ( ℃)
EPAS1	F：AAGACATGTCCACAGAGCGG R：TGTCGTCGCAGTAGGTGAAC	300	57.60 57.40
PPARα	F：GCGGCCCCAGGTGGT R：GATGCTGGATGACTCCTCGG	237	63.60 58.20
LC3B	F：CCGACTTATCCGAGAGCAGC R：TGAGCTGTAAGCGCCTTCTT	277	59.69 60.55
BNIP3	F：GAAGAACAGCTCCCAGTCCGA R：CCGACTGGACCAATCCCAA	103	61.77 59.32
NIX	F：ACTTAGAACAGCCGCCG R：CCACCCAGGAACTGTTGAGG	97	55.40 58.70
PINK1	F：TGTGGAACATCTCGGCAGG R：GGACCTCCCTTGGGTCTTCT	133	59.70 60.55
PRKN	F：CAGTGGCCATGATAGAACCTGA R：CAGAATCGACCTCCACTGGG	187	59.83 59.82
MFN1	F：AGACAGTTAATCAGCTGGCCC R：ATCTGTGCCCGGACTGTCTA	140	52.38 55.00
18sRNA	F：GTAACCCGTTGAACCCCATT R：CCATCCAATCGGTAGTAGCG	152	58.09 57.93

试剂 Reagent	15% 分离胶 15% resolving gel (mL)	5% 浓缩胶 5% stacking gel (mL)
H₂O	2.3	5.5
30% 丙烯酰胺溶液 30% Acr-Bis	5.0	1.3
Tris-HCl	2.5（pH 8.8，1.5 mol·L^-1）	1.0（pH 6.8，1.5 mol·L^-1）
10% 十二烷基硫酸钠 10% SDS	0.1	0.08
10% 过硫酸铵 10% APS	0.1	0.08
四甲基乙二胺 TEMED	0.004	0.008
总计 Total	10.0	8.0