藏绵羊肺脏结构特点及HIF-1α和AQP1的表达特性

doi:10.3864/j.issn.0578-1752.2023.11.013

摘要/Abstract

摘要：

【背景】低氧诱导因子-1α (hypoxia inducible factor-1α，HIF-1α) 是细胞对缺氧应激做出适应性反应的关键因子之一，主要通过调节基因转录来维持细胞中氧的供需平衡。水通道蛋白( Aquaporins，AQPs) 是疏水性的跨膜转运蛋白，为机体水稳态平衡提供重要的系统调节。肺中，AQP1主要通过调节肺泡、肺间质和毛细血管间水转运从而保持肺内液体平衡。高原环境由于低氧、高寒、大风和强辐射等特点，能够适应并生存的物种相对较少，藏羊作为适应高海拔、高寒气候的特有物种，形成了独特的形态结构和生理功能适应。肺脏作为呼吸主要的执行器官，对低氧或缺氧等极为敏感。【目的】通过研究藏绵羊肺脏结构特点以及HIF-1α与AQP1在不同海拔藏绵羊肺脏中的表达特性，揭示其在藏绵羊高原环境适应性中所起的作用。【方法】选择不同生存海拔藏绵羊和小尾寒羊，断颈致死后快速采集肺组织，H.E染色、PAS染色、Masson染色等观察肺脏显微结构及弹性纤维分布，免疫组织化学SP法及实时荧光定量PCR法检测肺组织中HIF-1α与AQP1蛋白及基因的表达特点。【结果】藏绵羊肺脏被膜厚度为40.28 μm，与小尾寒羊肺脏被膜厚度无显著差异，但其弹性纤维含量显著多于小尾寒羊（P<0.05）；藏绵羊细支气管黏膜上皮单位面积内杯状细胞数量显著多于小尾寒羊（P<0.05），但小支气管及以上分支中两者无显著差异；藏绵羊终末细支气管黏膜上皮仍有少量散在分布的杯状细胞且其终末细支气管平滑肌厚度显著高于小尾寒羊（P<0.05）；藏绵羊肺内微动脉（直径小于100 μm）平滑肌占血管外径比例显著小于小尾寒羊；藏绵羊呼吸性细支气管平滑肌厚度及单位面积内毛细血管数量显著高于小尾寒羊。肺脏中HIF-1α蛋白主要表达于细支气管黏膜上皮、肺微血管内皮和肺泡隔中，主要表达于细胞质；高海拔藏绵羊和小尾寒羊肺组织中HIF-1α表达均显著高于低海拔绵羊，低海拔藏绵羊肺脏中HIF-1α 的表达显著高于低海拔小尾寒羊。AQP1蛋白主要表达于肺泡上皮、肺泡隔及肺微血管内皮、细支气管平滑肌中，主要定位于细胞膜；高海拔藏绵羊肺组织中AQP1表达显著高于低海拔藏绵羊和小尾寒羊（P<0.05），但高海拔和低海拔小尾寒羊肺脏中AQP1的表达无显著差异（P>0.05）。【结论】藏绵羊肺被膜含有更多的弹性纤维，微血管平滑肌含量较少，但呼吸性细支气管上皮杯状细胞数量及平滑肌含量较多；藏绵羊肺脏中HIF-1α和AQP1的表达均显著高于小尾寒羊，且其表达均随海拔升高而增强。

关键词: 藏绵羊, HIF-1α, AQP1, 肺脏

Abstract:

【Background】HIF-1α is one of the key factors for cells to make adaptive response to hypoxia stress. It mainly maintains the balance of oxygen supply by regulating gene transcription. Aquaporins (AQPs) are hydrophobic transmembrane transporters regulating water homeostasis. Among them, AQP-1 mainly regulates the water transport among alveoli, pulmonary interstitium and capillaries, so as to maintain the fluid balance in the lung. The plateau environment is characterized by low oxygen, extremely cold, strong wind and radiation, so there are relatively few species which could adapt and survive. As the unique sheep species adapt to high altitude and high cold climate, Tibetan sheep has formed a special morphological structure and physiological functions adapted to the plateau environment. As the main executive organ of respiration, lung is very sensitive to hypoxia. 【Objective】 This study aimed to explore the structural characteristics of Tibetan sheep lung and the expression characteristics of HIF-1α and AQP1, so as to reveal the related roles of HIF-1α and AQP1 in Tibetan sheep high altitude adaption. 【Method】 The histochemical HE, PAS and Masson staining, immuno-histochemical SP and real-time fluorescence quantitative were used.【Result】The tunica thickness of lung in Tibetan sheep was 40.28 μm, which showed no significant difference with that of Small Tail Han sheep, but the elastic fiber proportion was significantly higher than that of Small Tail Han sheep (P<0.05); the number of goblet cells in bronchiolar epithelium in Tibetan sheep was significantly more than that in Small Tail Han sheep (P<0.05), but there was no significant difference in superior branches; some goblet cells were still detected in the epithelium of bronchioli terminales of Tibetan sheep; the thickness of smooth muscle of bronchioli terminales of Tibetan sheep was significantly thicker than that of Small Tail Han sheep (P<0.05); the proportion of smooth muscle in the pulmonary arteriole (diameter less than 100 μm) in Tibetan sheep was significantly less than that of Small Tail Han sheep; the thickness of respiratory bronchiole smooth muscle and the number of capillaries in Tibetan sheep were significantly higher than those in Small Tail Han sheep. The HIF-1α protein was mainly expressed in bronchiolar epithelium, pulmonary micro-vascular endothelium and alveolar septum, and mainly detected in cytoplasm; both Tibetan sheep and Small Tail Han sheep, the expression of HIF-1α was significantly higher in high altitude than that of low altitude, and HIF-1α expression in low altitude Tibetan sheep was significantly stronger than that of Small Tail Han sheep living at low altitude. AQP1 protein was mainly expressed in alveolar epithelium, alveolar septum, pulmonary microvascular endothelium, submucosal and smooth muscle of bronchioles, mainly located on cell membrane; the expression of AQP1 in lung of Tibetan sheep living at high altitude was significantly stronger than that of Tibetan sheep from low altitude, and also stronger than that of Small Tail Han sheep either altitude (P<0.05), but there was no significant difference between Small Tail Han sheep living at high altitude and low (P>0.05).【Conclusion】All those results indicated that the lung capsule of Tibetan sheep contained more elastic fibers and less microvascular smooth muscle than that of Small Tail Han sheep, with more number of goblet cells and smooth muscle in respiratory bronchioles. The expression of HIF-1α and AQP1 in Tibetan Sheep lung were significantly stronger than that of Small Tail Han sheep, and their expression increased with living altitude increasing.

Key words: Tibetan sheep, HIF-1α, AQP1, Lung

阿依木古丽·阿不都热依木, 张晨, 蔡勇, 覃圣, 罗文学, 扎西英派. 藏绵羊肺脏结构特点及HIF-1α和AQP1的表达特性[J]. 中国农业科学, 2023, 56(11): 2202-2211.

AYIMUGULI Abudureyimu, ZHANG Chen, CAI Yong, QIN Sheng, LUO WenXue, ZHAXIYINGPAI. The Micro-Structure of Tibetan Sheep Lung and Its HIF-1α and AQP1 Expression Characteristics[J]. Scientia Agricultura Sinica, 2023, 56(11): 2202-2211.

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引物名称 Primer name	序列号 Accession No.	序列 Sequence (5′-3′)	扩增长度 Amplicon size (bp)	退火温度 Tm (℃)
绵羊 β-Actin Sheep β-Actin	NM_001009784.3	F: GCAGATGTGGATCAGCAAGC R:TCTCGTTTTCTGCGCAAGTT	133	60
绵羊HIF-1α Sheep HIF-1α	XM_027971915.2	F: ATGTCTCCATTACCTGCCTCTGA R:CTGTTAGGCTCAGGTGAACTTTGT	197	56
绵羊AQP Sheep AQP	NM_001009194.1	F: ATCGTCGCCACTGTCATCCT R:TATCGCCAGCAGGTGTCCAA	240	58

	浆膜厚度 Tunica thickness (μm)	弹性纤维厚度 Elastic fibers thickness (μm)	弹性纤维百分比 Thickness proportion (%)
藏绵羊Tibetan sheep	40.28±1.58ns	21.95±1.65*	54.49*
小尾寒羊Small Tail Han sheep	39.24±2.15	12.25±1.55	31.21

	Reb平滑肌厚度 Smooth muscle of Reb (μm)	单个肺泡面积 APA (μm²)	肺泡隔毛细血管数 Capillary number (mm²)
藏绵羊 Tibetan sheep	25.12±1.25*	1256±56ns	113±12*
小尾寒羊 Small Tail Han sheep	8.05±0.75	1368±42	65±9

	肺微动脉直径 Pulmonary arteriole diameter (μm)
	<50	50—100	101—200	201—400
藏绵羊 Tibetan sheep	28.14±3.14a	22.32±5.14a	20.12±1.99a	18.24±3.21a
小尾寒羊 Small Tail Han sheep	36.36±2.36b	34.25±2.14b	21.24±2.24a	18.66±4.56a

	杯状细胞数Goblet cell numbers (mm²)		平滑肌厚度Smooth muscle thickness (μm)
	藏绵羊 Tibetan sheep	小尾寒羊 Small Tail Han sheep	藏绵羊 Tibetan sheep	小尾寒羊 Small Tail Han sheep
段支气管 Segmental bronchi	1854±98a	1759±65a	215.12±12.24a	204.26±14.35a
小支气管 Bronchium	1453±54a	1398±62a	175.14±4.13a	168.24±3.65a
细支气管 Bronchiole	541±24b	142±12c	45.34±2.55b	42.24±3.65b
终末细支气管 Bronchioli terminales	15±3d	ND	36.14±1.25b	21.09±2.34c