低氧习服在小鼠造血干细胞急性放射损伤修复中的作用观察

doi:10.3877/cma.j.issn.1673-9450.2023.04.004

中华损伤与修复杂志(电子版) ›› 2023, Vol. 18 ›› Issue (04) : 293 -298. doi: 10.3877/cma.j.issn.1673-9450.2023.04.004

论著

低氧习服在小鼠造血干细胞急性放射损伤修复中的作用观察

全勇, 冉新泽, 胡梦佳, 陈芳, 陈乃成, 廖伟年, 陈默, 申明强, 陈石磊, 王崧^†(

), 王军平^†(

)

400038　重庆，陆军军医大学（第三军医大学）军事预防医学系全军复合伤研究所　创伤、烧伤与复合伤国家重点实验室　重庆市纳米医学工程研究中心

收稿日期:2023-04-27 出版日期:2023-08-01
通信作者: 王崧, 王军平
基金资助:
国家自然科学基金重点项目(81930090); 军队后勤科研项目(ALJ19J002)

Observation of effect on hypoxia acclimatization in repair of hematopoietic stem cells after acute radiation injury in mice

Yong Quan, Xinze Ran, Mengjia Hu, Fang Chen, Naicheng Chen, Weinian Liao, Mo Chen, Mingqiang Shen, Shilei Chen, Song Wang^†(), Junping Wang^†()

Institute of Combined Injury of PLA, State Key Laboratory of Trauma, Burn and Combined Injury, College of Preventive Medicine, Army Medical University (Third Military Medical University), Chongqing Engineering Research Center of Nanomedicine, Chongqing 400038, China

Received:2023-04-27 Published:2023-08-01
Corresponding author: Song Wang, Junping Wang

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引用本文：

全勇, 冉新泽, 胡梦佳, 陈芳, 陈乃成, 廖伟年, 陈默, 申明强, 陈石磊, 王崧, 王军平. 低氧习服在小鼠造血干细胞急性放射损伤修复中的作用观察[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(04): 293-298.

Yong Quan, Xinze Ran, Mengjia Hu, Fang Chen, Naicheng Chen, Weinian Liao, Mo Chen, Mingqiang Shen, Shilei Chen, Song Wang, Junping Wang. Observation of effect on hypoxia acclimatization in repair of hematopoietic stem cells after acute radiation injury in mice[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2023, 18(04): 293-298.

目的

研究低氧习服在小鼠造血干细胞急性放射损伤后修复中的作用。

方法

采用随机数字表法将6~7周龄雄性C57BL/6J小鼠分为常氧辐照组和低氧习服辐照组。建立低氧习服急性放射损伤动物模型，低氧习服后再进行⁶⁰Co全身一次性照射6.0Gy。连续监测辐照前后小鼠尾静脉血血常规；流式细胞术检测低氧习服前后小鼠骨髓造血干细胞；取低氧习服小鼠长骨进行病理分析和VE-Cadherin免疫荧光染色；ELISA检测低氧习服小鼠低氧诱导因子-1α(HIF-1α)和血管生成素-1(ANGPT1)。

结果

低氧习服加速辐照后小鼠外周血恢复，低氧习服辐照组外周血红细胞和血小板最低值高于常氧辐照组[(7.0±0.6)×10¹²/L vs (5.1±0.4)×10¹²/L，t＝2.483，P<0.05；(203±30)×10⁹/L vs (141±25)×10⁹/L，t＝2.541，P<0.05]；低氧习服组骨髓LSK细胞和长期造血干细胞(LT-HSC)的比例及数量均高于常氧对照组[(3.91±0.21)‰ vs (3.37±0.11)‰，t＝2.536，P＝0.032；(7.18±0.84)×10⁴/L vs (6.01±0.82) ×10⁴/L，t＝2.641，P＝0.029；(12.05±0.53)% vs (9.37±0.45)%，t＝3.512，P＝0.023；(0.74±0.12)×10³/L vs (0.58±0.11)×10³/L，t＝2.667，P＝0.027]。低氧习服促进骨髓窦的增殖扩张和VE-Cadherin在窦内皮细胞的表达，减轻造血细胞凋亡和血管龛损伤；低氧习服组小鼠骨髓LSK细胞中HIF-1α和骨髓腔冲洗上清液中ANGPT1含量明显增加[(2.97±0.28)pg/cell vs (1.95±0.22)pg/cell，t＝4.896，P＝0.013；(12.30±3.70)pg/ml vs (6.25±2.70)pg/ml，t＝6.742，P＝0.001]。

结论

低氧习服减轻造血干细胞和血管龛的放射损伤，加快造血恢复，其原因可能与低氧习服调节LT-HSC比例，促进HIF-1α、ANGPT1在造血干细胞中的表达有关。

关键词: 低氧习服, 放射损伤, 造血干细胞, 低氧诱导因子-1α, 血管生成素-1

Objective

To investigate the effect of hypoxia acclimatization in repair of hematopoietic stem cells (HSCs) after acute radiation injury in mice.

Methods

According to random number table method, the male C57BL mice aged 6-7 weeks were divided into normoxic irradiation group and hypoxia acclimatization irradiation group. The animal model of acute radiation injury after hypoxia acclimatization was established by 6.0 Gy of one-time irradiation of ⁶⁰Co. Tail venous blood routine of mice before and after irradiation was continuously monitored. Detected the bone marrow hematopoietic stem cells in mice before and after hypoxia acclimatization by flow cytometry. Long bones of hypoxic acclimatized mice were taken for pathological analysis and VE-Cadherin immunofluorescence staining. HIF-1α and ANGPT1 were detected by ELISA in hypoxic acclimatized mice.

Results

Hypoxia acclimatization accelerated the recovery of peripheral blood after irradiation, the lowest numbers of RBC and PLT in hypoxia acclimatization irradiation group were higher than normoxic irradiation group [(7.0±0.6)×10¹²/L vs (5.1±0.4)×10¹²/L, t=2.483, P<0.05; (203±30)×10⁹/L vs (141±25)×10⁹/L, t=2.541, P<0.05]. The proportions and numbers of LSK cells and LT-HSC in the bone marrow of hypoxia acclimatization group were significantly higher than normoxic control group[(3.91±0.21)‰ vs (3.37±0.11) ‰, t=2.536, P=0.032; (7.18±0.84)×10⁴/L vs (6.01±0.82)×10⁴/L, t=2.641, P=0.029; (12.05±0.53)% vs (9.37±0.45) %, t=3.512, P=0.023; (0.74±0.12)× 10³/L vs (0.58±0.11) ×10³/L, t=2.667, P=0.027]. Hypoxia acclimatization caused proliferation and expansion of the bone marrow sinuses, and strengthened the expression of VE-Cadherin in endothelial sinusoidal cells. It also alleviated hematopoietic cell apoptosis and vascular niche damage. HIF-1α in LSK cells and ANGPT1 in the bone marrow cavity flushing supernatant increased significantly[(2.97±0.28)pg/cell vs (1.95±0.22)pg/cell, t=4.896, P=0.013; (12.30±3.70)pg/ml vs (6.25±2.70)pg/ml, t=6.742, P=0.001].

Conclusion

Hypoxia acclimatization attenuated the injuries and accelerated the repair of HSC induced by radiation. It may be related to the fact that hypoxia acclimatization regulate the long-term hematopoietic stem cell ratio, promote the expression of HIF-1α and ANGPT1 in HSC.

Key words: Hypoxic acclimatization, Radiation injury, HSC, HIF-1α, ANGPT1

图1 辐照后外周血常规变化。A示外周血白细胞计数；B示外周血红细胞计数；C示外周血血小板计数。与常氧辐照组相比，^*P<0.05

图2 6.0 Gy辐照后小鼠股骨骨髓组织病理变化(HE，400×)。与相同时间点常氧辐照组比较，低氧习服辐照组小鼠骨髓血管系统尤其是骨髓窦扩张及破裂程度更轻，骨髓内出血也相对更少。黄色箭头所指为骨髓窦。A示常氧辐照组；B示低氧习服辐照组

表1 低氧习服对小鼠骨髓LSK和LT－HSC比例及数量的影响(±s)

组别	LSK比例(‰)	LT－HSC占LSK细胞比例(%)	LSK细胞数量(×10⁴/L)	LT－HSC数量(×10³/L)
常氧对照组	3.37±0.11	9.37±0.45	6.01±0.82	0.58±0.11
低氧习服组	3.91±0.21	12.05±0.53	7.18±0.84	0.74±0.12
t值	2.536	3.512	2.641	2.667
P值	0.032	0.023	0.029	0.027

表1 低氧习服对小鼠骨髓LSK和LT－HSC比例及数量的影响(±s)

组别	LSK比例(‰)	LT－HSC占LSK细胞比例(%)	LSK细胞数量(×10⁴/L)	LT－HSC数量(×10³/L)
常氧对照组	3.37±0.11	9.37±0.45	6.01±0.82	0.58±0.11
低氧习服组	3.91±0.21	12.05±0.53	7.18±0.84	0.74±0.12
t值	2.536	3.512	2.641	2.667
P值	0.032	0.023	0.029	0.027

图3 流式细胞术分析小鼠骨髓造血干细胞LSK和LT-HSC。A示LSK；B示LT-HSC

图4 骨髓窦及VE-Cadherin表达变化(400×)。低氧习服小鼠骨髓窦增加，窦腔明显扩张，小动脉及骨髓窦显强绿色荧光，巨核细胞及红细胞自发弱绿色荧光。A示常氧对照组，小鼠骨髓窦管腔小，VE-Cadherin表达弱；B示低氧习服组，小鼠骨髓窦环周VE-Cadherin表达明显增强。黄色箭头所指为骨髓窦

表2 小鼠骨髓LSK细胞中HIF－1α和骨髓腔冲洗上清液ANGPT1含量(±s)

组别	HIF－1α(pg/cell)	ANGPT1(pg/ml)
常氧对照组	1.95±0.22	6.25±2.70
低氧习服组	2.97±0.28	12.30±3.70
t值	4.896	6.742
P值	0.013	0.001

表2 小鼠骨髓LSK细胞中HIF－1α和骨髓腔冲洗上清液ANGPT1含量(±s)

组别	HIF－1α(pg/cell)	ANGPT1(pg/ml)
常氧对照组	1.95±0.22	6.25±2.70
低氧习服组	2.97±0.28	12.30±3.70
t值	4.896	6.742
P值	0.013	0.001

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