山羊脐带间充质干细胞分离培养与治疗小鼠药物性肝损伤

doi:10.11843/j.issn.0366-6964.2025.08.042

摘要/Abstract

摘要：

本研究旨在建立雷州山羊脐带间充质干细胞(LZG-UCMSCs)的分离培养体系，并探讨其治疗小鼠药物性肝损伤(DILI)的效果。选用9日龄的雷州山羊进行脐带间充质干细胞(UCMSCs)的分离培养，并开展生长曲线检测、细胞克隆、RT-PCR、免疫荧光和诱导试验，检验分离的细胞是否为UCMSCs。将36只ICR小鼠随机分为3组：空白对照组、模型损伤组、干细胞治疗组，每组12只。模型组和治疗组按照15 mg·kg^-1的剂量对小鼠进行异烟肼灌胃，持续4周，然后检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和碱性磷酸酶(ALP)水平。结果表明，分离培养的LZG-UCMSCs的细胞形态正常，具有UCMSCs的特性，与对照组相比，损伤组ALT、AST和ALP水平显著升高(P < 0.05)；与损伤组相比，治疗组ALT、AST和ALP水平极显著降低(P < 0.01)。结果提示，可以从雷州山羊脐带中分离出正常的UCMSCs，LZG-UCMSCs对小鼠DILI有一定的治疗作用，研究结果对LZG-UCMSCs进一步治疗山羊同源DILI具有一定参考价值。

关键词: 山羊, 脐带, 间充质干细胞, 小鼠, 肝损伤, 治疗

Abstract:

The aim of this study was to observe the isolation and culture of Leizhou goat umbilical cord mesenchymal stem cells (LZG-UCMSCs) and the effect on the treatment of drug-induced liver injury (DILI) in mice. Umbilical cord mesenchymal stem cells were isolated and cultured, growth curve, cell cloning, RT-PCR, immunofluorescence and induction experiments were performed from nine-year-old Leizhou goat placenta. 36 ICR mice were randomly divided into 3 groups: blank control group, model injury group and stem cell treatment group, with 12 mice in each group. The rats in the model group and the treatment group were given gavage at a dose of 15 mg·kg^-1 for 4 weeks. Aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were measured. The results showed that the cell morphology of the isolated and cultured LZG-UCMSCs was normal, with the characteristics of UCMSCs. Compared with the control group, the activities of ALT, AST and ALP in the injured group were significantly increased (P < 0.05). Compared with injury group, ALT, AST and ALP in treatment group were significantly decreased (P < 0.01). The results suggest that normal UCMSCs can be isolated from the placenta of Lezhou goat, and LZG-UCMSCs has a certain therapeutic effect on DILI in mice, thus providing reference value for further treatment of homologous goat DILI by LZG-UCMSCs.

Key words: goat, umbilical cord, mesenchymal stem cells, mouse, liver injury, treatment

中图分类号:

S856.4

冯锦涛, 闫炎, 宗俊霖, 胡宇晴, 李晓蕊, 关伟军, 李耀坤. 山羊脐带间充质干细胞分离培养与治疗小鼠药物性肝损伤[J]. 畜牧兽医学报, 2025, 56(8): 4053-4064.

FENG Jintao, YAN Yan, ZONG Junlin, HU Yuqing, LI Xiaorui, GUAN Weijun, LI Yaokun. Isolation and Culture of Goat Umbilical Cord Mesenchymal Stem Cells for the Treatment of Drug-induced Liver Injury in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 4053-4064.

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参考文献 33

1	MAO A S , MOONEY D J . Regenerative medicine: current therapies and future directions[J]. Proc Natl Acad Sci USA, 2015, 112 (47): 14452- 14459. doi: 10.1073/pnas.1508520112
2	龚业莉. 走近神奇的干细胞移植[M]. 西安: 西安交通大学出版社, 2014.
	GONG Y L . Approaching the magical stem cell transplantation[M]. Xi'an: Xi'an Jiaotong University Press, 2014.
3	陈雁雁, 熊御云, 郑允文, 等. 干细胞与再生医学[J]. 皮肤科学报, 2024, 41 (5): 469-472, 478.
	CHEN Y Y , XIONG Y Y , ZHENG Y W , et al. Stem cells and regenerative medicine[J]. Journal of Dermatology, 2024, 41 (5): 469-472, 478.
4	WANG J P , DENG G , WANG S Y , et al. Enhancing regenerative medicine: the crucial role of stem cell therapy[J]. Front Neurosci, 2024, 18, 1269577. doi: 10.3389/fnins.2024.1269577
5	王佃亮. 脐带间充质干细胞的发现研究及规模化生产——《脐带间充质干细胞》连载之一[J]. 中国生物工程志, 2018, 38 (8): 108- 112.
	WANG D L . Discovery, research and large-scale production of umbilical cord mesenchymal stem cells-serial one of "Umbilical cord mesenchymal stem cells"[J]. Journal of Bioengineering in China, 2018, 38 (8): 108- 112.
6	张冬林. miR-122修饰的脐带血间充质干细胞对慢加急性肝衰竭大鼠的作用分析[D]. 太原: 山西医科大学, 2022.
	ZHANG D L. Analysis of the effect of Mir-122-modified umbilical cord blood mesenchymal stem cells on rats with acute-on-chronic liver failure[D] Taiyuan: Shanxi Medical University, 2022. (in Chinese)
7	袁吉祥, 魏述军, 范长青. 人脐带血间充质干细胞磁靶向移植对急性心肌梗死再灌注模型心功能的影响[J]. 宁夏医学杂志, 2019, 41 (8): 687-690, 672.
	YUAN J X , WEI S J , FAN C Q . The effect of magnetic targeted transplantation of human umbilical cord blood mesenchymal stem cells on cardiac function in acute myocardial infarction reperfusion models[J]. Ningxia Medical Journal, 2019, 41 (8): 687-690, 672.
8	杨汉华, 田树凤, 谢丽春, 等. 经鼻腔移植人脐带间充质干细胞修复新生大鼠缺氧缺血性脑损伤[J]. 中国组织工程研究, 2019, 23 (21): 3386- 3391.
	YANG H H , TIAN S F , XIE L C , et al. Nasal transplantation of human umbilical cord mesenchymal stem cells for the repair of hypoxic-ischemic brain injury in neonatal rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23 (21): 3386- 3391.
9	俞静. 人脐带间质干细胞外泌体对肾间质纤维化的修复及机制研究[D]. 镇江: 江苏大学, 2017.
	YU J. Research on the repair and mechanism of human umbilical cord mesenchymal stem cell exosomes in renal interstitial fibrosis[D]. Zhenjiang: Jiangsu University, 2017. (in Chinese)
10	金梓渊, 周颖, 潘小平, 等. 脂肪间充质干细胞来源的胞外囊泡治疗急慢性肝损伤研究进展[J]. 中西医结合肝病杂志, 2024, 34 (8): 764- 768.
	JIN Z Y , ZHOU Y , PAN X P , et al. Research progress on extracellular vesicles derived from adipose-derived mesenchymal stem cells in the treatment of acute and chronic liver injury[J]. Journal of Integrated Traditional Chinese and Western Medicine on Hepatology, 2024, 34 (8): 764- 768.
11	耿江苏, 刘翠艳. 肝损伤对畜禽的危害[C]//中国畜牧兽医学会. 创新、融合、健康、未来—第九届全国畜牧兽医青年科技工作者学术研讨会论文集. 重庆, 2020: 345.
	GENG J S, LIU C Y. The hazards of liver injury to livestock and poultry[C]// Chinese Veterinary Medical Association. Innovation, integration, health, future-Proceedings of the 9th National Academic Symposium on Young Science and Technology Workers in Animal Husbandry and Veterinary Medicine. Chongling, 2020: 345. (in Chinese)
12	周鸿飞. 牛磺酸对大肠杆菌感染致肉鸡肝损伤的保护作用[D]. 沈阳: 沈阳农业大学, 2022.
	ZHOU H F. The protective effect of taurine on liver injury in broilers caused by Escherichia coli infection[D]. Shenyang: Shenyang Agricultural University, 2022. (in Chinese)
13	龚倩梅, 王晓明, 王丽平. 大蒜辣素对CCl4诱导的小鼠急性肝损伤的保护作用研究[C]//中国毒理学会. 中国毒理学会第十一次全国毒理学大会论文集. 苏州, 2024: 225-226.
	GONG Q M, WANG X M, WANG L P. Study on the protective effect of allicin on CCL4-induced acute liver injury in mice[C]// Chinese Society of Toxicology. Proceedings of the 11th National Toxicology Conference of the Chinese Society of Toxicology. Suzhou, 2024: 225-226. (in Chinese)
14	文晴, 杨梦娇, 原玉柱, 等. 中性粒细胞在药物性肝损伤中的作用研究进展[J]. 药学进展, 2024, 48 (10): 796- 802.
	WEN Q , YANG M J , YUAN Y Z , et al. Research progress on the role of neutrophils in drug-induced liver injury[J]. Pharmaceutical Progress, 2024, 48 (10): 796- 802.
15	杜佳欣, 李巴仑, 寇正, 等. 血清饥饿法在间充质干细胞治疗犬糖尿病中的应用(英文)[J]. 农业生物技术学报, 2024, 32 (12): 2773- 2785.
	DU J X , LI B L , KOU Z , et al. Application of serum hunger method in mesenchymal stem cell therapy for canine diabetes (English)[J]. Transactions of the Chinese Society of Agricultural Biotechnology, 2024, 32 (12): 2773- 2785.
16	王福军. 干细胞因子在3例犬急性脊髓神经系统疾病治疗中的应用和体会[J]. 中国兽医杂志, 2024, 60 (4): 138- 142.
	WANG F J . Application and experience of stem cell factors in the treatment of three cases of acute spinal neurological diseases in dogs[J]. Chinese Journal of Veterinary Medicine, 2024, 60 (4): 138- 142.
17	王凯. 高活性间充质干细胞治疗猕猴卵巢衰老的细胞和分子机制研究[D]. 昆明: 昆明医科大学, 2023.
	WANG K. Research on the cellular and molecular mechanisms of high-activity mesenchymal stem cell therapy for ovarian aging in macaques[D]. Kunming: Kunming Medical University, 2023. (in Chinese)
18	宗俊霖, 王亚芹, 刘晏辰, 等. 荷斯坦牛脐带间充质干细胞分离鉴定及分化研究[J]. 中国农业科技导报, 2023, 25 (9): 105- 112.
	ZONG J L , WANG Y Q , LIU Y C , et al. Isolation, identification and differentiation study of umbilical cord mesenchymal stem cells from Holstein cattle[J]. China Agricultural Science and Technology Review, 2023, 25 (9): 105- 112.
19	周世莹, 刘晏辰, 张洋, 等. 大耳白兔骨髓间充质干细胞的分离培养与生物学鉴定[J]. 中国农业科技导报, 2023, 25 (8): 96- 105.
	ZHOU S Y , LIU Y C , ZHANG Y , et al. Isolation, culture and biological identification of bone marrow mesenchymal stem cells from White rabbits with large ears[J]. China Agricultural Science and Technology Review, 2023, 25 (8): 96- 105.
20	赵善民, 汪芸, 刘畅, 等. 常用药物性肝损伤动物模型的制备方法[J]. 中国实验动物学报, 2023, 31 (7): 935- 945.
	ZHAO S M , WANG Y , LIU C , et al. Preparation methods of common animal models of drug-induced liver injury[J]. Chinese Journal of Laboratory Animal Science, 2023, 31 (7): 935- 945.
21	黄淼. 三维培养对间充质干细胞特性及其成骨分化的影响研究[D]. 赣州: 赣南医科大学, 2024.
	HUANG M. Research on the effects of three-dimensional culture on the characteristics of mesenchymal stem cells and their osteogenic differentiation[D]. Ganzhou: Gannan Medical University, 2024. (in Chinese)
22	杨刚, 李永继, 熊永福, 等. PON1基因低表达对肝癌细胞增殖和细胞克隆能力的影响[J]. 重庆医学, 2023, 52 (22): 3391- 3396. doi: 10.3969/j.issn.1671-8348.2023.22.005
	YANG G , LI Y J , XIONG Y F , et al. The effect of low expression of PON1 gene on the proliferation and cell cloning ability of hepatoma cells[J]. Chongqing Medical Journal, 2023, 52 (22): 3391- 3396. doi: 10.3969/j.issn.1671-8348.2023.22.005
23	楼梦雨, 潘欠欠, 敬敬, 等. 绵羊骨髓间充质干细胞分离培养与鉴定[J]. 中国兽医学报, 2022, 42 (11): 2299- 2304.
	LOU M Y , PAN Q Q , JING J , et al. Isolation, culture and identification of sheep bone marrow mesenchymal stem cells[J]. Chinese Journal of Veterinary Medicine, 2022, 42 (11): 2299- 2304.
24	翟竹汇. 猫脐带间充质干细胞的分离鉴定及NF-κB通路在其抗氧化应激中的作用研究[D]. 太谷: 山西农业大学, 2023.
	ZHAI Z H. Isolation and identification of cat umbilical cord mesenchymal Stem cells and Study on the role of NF-κB Pathway in their antioxidant Stress[D] Taigu: Shanxi Agricultural University, 2023. (in Chinese)
25	马永杰, 赵鹏鹏, 龚素华, 等. 秦岭大熊猫骨髓、牙髓和脂肪组织来源间充质干细胞的分离与鉴定[J]. 动物医学进展, 2023, 44 (10): 24- 30.
	MA Y J , ZHAO P P , GONG S H , et al. Isolation and identification of mesenchymal stem cells derived from bone marrow, dental pulp and adipose tissue of Qinling Giant Pandas[J]. Advances in Veterinary Medicine, 2023, 44 (10): 24- 30.
26	DELORENZIL P R . Neocryptic karyotype difference between cattle and goats[J]. Chromosomes, 2015, 23 (2): 225- 235. doi: 10.1007/s10577-014-9462-4
27	PANCHAL N K , SWARNAL ATHA P , PRINCE S E . Trichopus zeylanicus ameliorates ibuprofen inebriated hepatotoxicity and enteropathy: an insight into its modulatory impact on pro/anti-inflammatory cytokines and apoptotic signaling pathways[J]. Inflammopharmacology, 2022, 30 (6): 2229- 2242. doi: 10.1007/s10787-022-01052-5
28	魏媛媛, 樊艺萌, 袁燕燕, 等. 茵山莲水提物对四氯化碳诱导小鼠急性肝损伤的保护作用[J]. 畜牧兽医学报, 2022, 53 (7): 2333- 2342. doi: 10.11843/j.issn.0366-6964.2022.07.030
	WEI Y Y , FAN Y M , YUAN Y Y , et al. Protective effect of aqueous extract of Yinshanlian on acute liver injury induced by carbon tetrachloride in mice[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (7): 2333- 2342. doi: 10.11843/j.issn.0366-6964.2022.07.030
29	AITHAL G P , WATKINS P B , ANDRADE R J , et al. Case definition and phenotype standardization in drug-induced liver injury[J]. Clin Pharmacol Ther, 2011, 89 (6): 806- 815. doi: 10.1038/clpt.2011.58
30	王超, 蔡华俊, 王晨晖, 等. 网络药理学研究筒鞘蛇菰治疗药源性肝损伤的机制[J]. 华西药学杂志, 2024, 39 (5): 515- 521.
	WANG C , CAI H J , WANG C H , et al. Network pharmacology study on the mechanism of Balanophora japonicum in the treatment of drug-induced liver injury[J]. West China Journal of Pharmacy, 2024, 39 (5): 515- 521.
31	王燕, 陈翠青, 徐岩, 等. 五味保肝丸对非酒精性脂肪性肝病小鼠的干预作用及机制[J]. 中国药房, 2024, 35 (11): 1345- 1350.
	WANG Y , CHEN C Q , XU Y , et al. The intervention effect and mechanism of Wuwei Baogan Pills on mice with non-alcoholic fatty liver disease[J]. Chinese Pharmacy, 2024, 35 (11): 1345- 1350.
32	李明远, 张玮, 华梦晴. 甲基巴多索龙通过抑制NLRP3炎症小体活化缓解小鼠急性肝损伤[J]. 南方医科大学学报, 2024, 44 (9): 1662- 1669.
	LI M Y , ZHANG W , HUA M Q . Methylbardoxolone alleviates acute liver injury in mice by inhibiting the activation of NLRP3 inflammasome[J]. Journal of Southern Medical University, 2024, 44 (9): 1662- 1669.
33	白鲁源, 孙光碧, 杨丽, 等. 柴芍六君子汤对酒精性肝损伤小鼠的治疗作用及机制[J]. 精准医学杂志, 2024, 39 (3): 242- 246.
	BAI L Y , SUN G B , YANG L , et al. The therapeutic effect and mechanism of Chai Shao Liu Jun Zi Decoction on mice with alcoholic liver injury[J]. Journal of Precision Medicine, 2024, 39 (3): 242- 246.

基因 Gene	引物序列(5′→3′) Sequence	片段长度/bp Product length
GAPDH	F：GGCGTGAACCACGAGAAGTA	141
	R：GGCGTGGACAGTGGTCATAA
CD29	F：GAATCGGGTTTGGCTCTTTCGT	309
	R：AATGGGCTCGTGCAGTTCTGTT
CD45	F：GATCCACCCCCAACCTCATCT	196
	R：AACTGCAAGAATCAAAGCCA
CD73	F：TGGGAGGAATTCACGTGGTG	104
	R：AGGCTCATAACTGGGCACAC
CD90	F：CTGACCCGTGATACAAAGAAGTGG	216
	R：CCTTGTTGGAGACGGTGGGATT
CD105	F：TGCACCATCATAAGCCTGACCTT	136
	R：TGACCACCTCGTTACTGACCACAT

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